bluuebunny/crossref_metadata_2025 huggingface dataset:
Insertion or Deletion of the Cheo Box Modifies Radiation Inducibility of
Clostridium
Promoters
Authors: Nuyts S. , Van Mellaert L. , Barbé S. , Lammertyn E. , Theys J. , Landuyt W. , Bosmans E. , Lambin P. , Anné J.
Published: July 2002

Radiation-inducible promoters are being used in many viral vector systems to obtain spatial and temporal control of gene expression. It was previously proven that radiation-induced gene expression can also be obtained in a bacterial vector system using anaerobic apathogenic clostridia. The effect of radiation inducibility was detected using mouse tumor necrosis factor alpha (mTNF-α) as a model protein under regulation of the radiation-inducible
recA
promoter. In this report, experiments are described in which this
recA
promoter was modified in order to increase radiation responsiveness. Incorporation of an extra Cheo box in the
recA
promoter region resulted in an increase in mTNF-α secretion from 44% for the wild-type promoter to 412% for the promoter with an extra Cheo box after a single irradiation dose of 2 Gy. Deletion of the Cheo box in the promoter region eliminated radiation inducibility. These results prove that the Cheo box in the
recA
promoter is indeed the radiation-responsive element. We also tested whether we could induce the constitutive endo-β-1,4-glucanase promoter (
eglA
) via ionizing irradiation by introducing a Cheo box in the promoter region. While the use of the constitutive promoter did not lead to an increase in mTNF-α secretion after irradiation, the introduction of a Cheo box resulted in a 242% increase in mTNF-α secretion. Reverse transcriptase PCR of RNA samples isolated from irradiated and nonirradiated bacterial cultures demonstrated that the increase in secretion was the result of enhanced transcription of the mTNF-α gene.
Generation of Metal-Binding Staphylococci through Surface Display of Combinatorially Engineered Cellulose-Binding Domains
Authors: Wernérus Henrik , Lehtiö Janne , Teeri Tuula , Nygren Per-Åke , Ståhl Stefan
Published: July 2002

Ni
2+
-binding staphylococci were generated through surface display of combinatorially engineered variants of a fungal cellulose-binding domain (CBD) from
Trichoderma reesei
cellulase Cel7A. Novel CBD variants were generated by combinatorial protein engineering through the randomization of 11 amino acid positions, and eight potentially Ni
2+
-binding CBDs were selected by phage display technology. These new variants were subsequently genetically introduced into chimeric surface proteins for surface display on
Staphylococcus carnosus
cells. The expressed chimeric proteins were shown to be properly targeted to the cell wall of
S. carnosus
cells, since full-length proteins could be extracted and affinity purified. Surface accessibility for the chimeric proteins was demonstrated, and furthermore, the engineered CBDs, now devoid of cellulose-binding capacity, were shown to be functional with regard to metal binding, since the recombinant staphylococci had gained Ni
2+
-binding capacity. Potential environmental applications for such tailor-made metal-binding bacteria as bioadsorbents in biofilters or biosensors are discussed.
Variation in Resistance to High Hydrostatic Pressure and
rpoS
Heterogeneity in Natural Isolates of
Escherichia coli
O157:H7
Authors: Robey Marianne , Benito Amparo , Hutson Roger H. , Pascual Cristina , Park Simon F. , Mackey Bernard M.
Published: July 2002

Several natural isolates of
Escherichia coli
O157:H7 have previously been shown to exhibit stationary-phase-dependent variation in their resistance to inactivation by high hydrostatic pressure. In this report we demonstrate that loss of the stationary-phase-inducible sigma factor RpoS resulted in decreased resistance to pressure in
E. coli
O157:H7 and in a commensal strain. Furthermore, variation in the RpoS activity of the natural isolates of O157:H7 correlated with the pressure resistance of those strains. Heterogeneity was noted in the
rpoS
alleles of the natural isolates that may explain the differences in RpoS activity. These results are consistent with a role for
rpoS
in mediating resistance to high hydrostatic pressure in
E. coli
O157:H7.
Denaturing Gradient Gel Electrophoresis Analysis of the 16S rRNA Gene V1 Region To Monitor Dynamic Changes in the Bacterial Population during Fermentation of Italian Sausages
Authors: Cocolin Luca , Manzano Marisa , Cantoni Carlo , Comi Giuseppe
Published: July 2002

In this study, a PCR-denaturing gradient gel electrophoresis (DGGE) protocol was used to monitor the dynamic changes in the microbial population during ripening of natural fermented sausages. The method was first optimized by using control strains from international collections, and a natural sausage fermentation was studied by PCR-DGGE and traditional methods. Total microbial DNA and RNA were extracted directly from the sausages and subjected to PCR and reverse transcription-PCR, and the amplicons obtained were analyzed by DGGE. Lactic acid bacteria (LAB) were present together with other organisms, mainly members of the family
Micrococcaceae
and meat contaminants, such as
Brochothrix thermosphacta
and
Enterococcus
sp., during the first 3 days of fermentation. After 3 days, LAB represented the main population, which was responsible for the acidification and proteolysis that determined the characteristic organoleptic profile of the Friuli Venezia Giulia fermented sausages. The PCR-DGGE protocol for studying sausage fermentation proved to be a good tool for monitoring the process in real time, and it makes technological adjustments possible when they are required.
Toxicity Analysis of N- and C-Terminus-Deleted Vegetative Insecticidal Protein from
Bacillus thuringiensis
Authors: Selvapandiyan A. , Arora N. , Rajagopal R. , Jalali S. K. , Venkatesan T. , Singh S. P. , Bhatnagar Raj K.
Published: July 2002

A vegetative insecticidal protein (VIP)-encoding gene from a local isolate of
Bacillus thuringiensis
has been cloned, sequenced, and expressed in
Escherichia coli
. The expressed protein shows insecticidal activity against several lepidopteran pests but is ineffective against
Agrotis ipsilon
. Comparison of the amino acid sequence with those of reported VIPs revealed a few differences. Analysis of insecticidal activity with N- and C-terminus deletion mutants suggests a differential mode of action of VIP against different pests.
Involvement of Nitrate Reductase and Pyoverdine in Competitiveness of
Pseudomonas fluorescens
Strain C7R12 in Soil
Authors: Mirleau Pascal , Philippot Laurent , Corberand Thérèse , Lemanceau Philippe
Published: July 2002

Involvement of nitrate reductase and pyoverdine in the competitiveness of the biocontrol strain
Pseudomonas fluorescens
C7R12 was determined, under gnotobiotic conditions, in two soil compartments (bulk and rhizosphere soil), with the soil being kept at two different values of matric potential (−1 and −10 kPa). Three mutants affected in the synthesis of either the nitrate reductase (Nar
−
), the pyoverdine (Pvd
−
), or both (Nar
−
Pvd
−
) were used. The Nar
−
and Nar
−
Pvd
−
mutants were obtained by site-directed mutagenesis of the wild-type strain and of the Pvd
−
mutant, respectively. The selective advantage given by nitrate reductase and pyoverdine to the wild-type strain was assessed by measuring the dynamic of each mutant-to-total-inoculant (wild-type strain plus mutant) ratio. All three mutants showed a lower competitiveness than the wild-type strain, indicating that both nitrate reductase and pyoverdine are involved in the fitness of
P. fluorescens
C7R12. The double mutant presented the lowest competitiveness. Overall, the competitive advantages given to C7R12 by nitrate reductase and pyoverdine were similar. However, the selective advantage given by nitrate reductase was more strongly expressed under conditions of lower aeration (−1 kPa). In contrast, the selective advantage given by nitrate reductase and pyoverdine did not differ in bulk and rhizosphere soil, indicating that these bacterial traits are not specifically involved in the rhizosphere competence but rather in the saprophytic ability of C7R12 in soil environments.
Inoculation onto Solid Surfaces ProtectsSalmonellaspp. during Acid Challenge: a Model Study Using Polyethersulfone Membranes
Authors: Gawande Purushottam V. , Bhagwat Arvind A.
Published: July 2002

Salmonellae are the most frequently reported cause of outbreaks of food-borne gastroenteritis in the United States. In clinical trials, the oral infective dose (ID) for healthy volunteers was estimated to be approximately 1 million cells. However, in reports from various outbreaks, the ID ofSalmonellaspecies associated with solid foods was estimated to be as few as 100 cells. We found that fresh-cut produce surfaces not only provided suitable solid support for pathogen attachment but also played a critical role in increasing the acid tolerance of the pathogen. However the acidic nature of certain produce played no role in making salmonellae resistant to stomach acidity. Inoculation onto fresh-cut produce surfaces, as well as onto inert surfaces, such as polyethersulfone membranes and tissue paper, increased the survival of salmonellae during acid challenge (50 mM Na-citrate, pH 3.0; 37°C; 2 h) by 4 to 5 log units. Acid challenge experiments using cells inoculated onto polyethersulfone membranes provided a model system suitable for studying the underlying fundamentals of the protection that occurs whenSalmonellastrains are associated with solid foods. The surface-associated acid protection, which was observed in severalSalmonellastrains, required de novo protein synthesis and was independent of stationary-phase sigma transcription factor.
Higher Abundance of Bacteria than of Viruses in Deep Mediterranean Sediments
Authors: Danovaro Roberto , Manini Elena , Dell'Anno Antonio
Published: July 2002

The interactions between viral abundance and bacterial density, biomass, and production were investigated along a longitudinal transect consisting of nine deep-sea stations encompassing the entire Mediterranean basin. The numbers of viruses were very low (range, 3.6 × 10
7
to 12.0 × 10
7
viruses g
−1
) and decreased eastward. The virus-to-bacterium ratio was always < 1.0, indicating that the deep-sea sediments of the Mediterranean Sea are the first example of a marine ecosystem not numerically dominated by viruses. The lowest virus numbers were found where the lowest bacterial metabolism and turnover rates and the largest cell size were observed, suggesting that bacterial doubling time might play an important role in benthic virus development.
Cre-
loxP
Recombination System for Large Genome Rearrangements in
Lactococcus lactis
Authors: Campo Nathalie , Daveran-Mingot Marie-Line , Leenhouts Kees , Ritzenthaler Paul , Le Bourgeois Pascal
Published: July 2002

We have used a new genetic strategy based on the Cre-
loxP
recombination system to generate large chromosomal rearrangements in
Lactococcus lactis
. Two
loxP
sites were sequentially integrated in inverse order into the chromosome either at random locations by transposition or at fixed points by homologous recombination. The recombination between the two chromosomal
loxP
sites was highly efficient (approximately 1 × 10
−1
/cell) when the Cre recombinase was provided in
trans
, and parental- or inverted-type chromosomal structures were isolated after removal of the Cre recombinase. The usefulness of this approach was demonstrated by creating three large inversions of 500, 1,115, and 1,160 kb in size that modified the lactococcal genome organization to different extents. The Cre-
loxP
recombination system described can potentially be used for other gram-positive bacteria without further modification.
Identification of Novel Hexapeptides Bioactive against Phytopathogenic Fungi through Screening of a Synthetic Peptide Combinatorial Library
Authors: López-García Belén , Pérez-Payá Enrique , Marcos Jose F.
Published: July 2002

The purpose of the present study was to improve the antifungal activity against selected phytopathogenic fungi of the previously identified hexapeptide PAF19. We describe some properties of a set of novel synthetic hexapeptides whosed-amino acid sequences were obtained through screening of a synthetic peptide combinatorial library in a positional scanning format. As a result of the screening, 12 putative bioactive peptides were identified, synthesized, and assayed. The peptides PAF26 (Ac-rkkwfw-NH2), PAF32 (Ac-rkwhfw-NH2), and PAF34 (Ac-rkwlfw-NH2) showed stronger activity than PAF19 against isolates ofPenicillium digitatum,Penicillium italicum, andBotrytis cinerea. PAF26 and PAF32, but not PAF34, were also active againstFusarium oxysporum. Penicillium expansumwas less susceptible to all four PAF peptides, and only PAF34 showed weak activity against it. Assays were also conducted on nontarget organisms, and PAF26 and PAF32 showed much-reduced toxicity toEscherichia coliandSaccharomyces cerevisiae,demonstrating selectivity towards certain filamentous fungi. Thus, the data showed distinct activity profiles for peptides differentiated by just one or two residue substitutions. Our conclusion from this observation is that a specificity factor is involved in the activity of these short peptides. Furthermore, PAF26 and PAF32 displayed activities againstP. digitatum,P. italicum, andB. cinereasimilar to that of the hemolytic 26-amino acid melittin, but they did not show the high toxicity of melittin towards bacteria and yeasts. The four peptides acted additively, with no synergistic interactions among them, and PAF26 was shown to have improved activity over PAF19 in in vivo orange fruit decay experiments.
Alterations in
Vibrio fischeri
Motility Correlate with a Delay in Symbiosis Initiation and Are Associated with Additional Symbiotic Colonization Defects
Authors: Millikan Deborah S. , Ruby Edward G.
Published: July 2002

Motility is required for
Vibrio fischeri
cells to interact with and specifically colonize the light-emitting organ of their host, the squid
Euprymna scolopes
. To investigate the influence of motility on the expression of the symbiotic phenotype, we isolated mutants of the squid symbiont
V. fischeri
ES114 that had altered migration abilities. Spontaneous hyperswimmer (HS) mutants, which migrated more rapidly in soft agar and were hyperflagellated relative to the wild type, were isolated and grouped into three phenotypic classes. All of the HS strains tested, regardless of class, were delayed in symbiosis initiation. This result suggested that the hypermotile phenotype alone contributes to an inability to colonize squid normally. Class III HS strains showed the greatest colonization defect: they colonized squid to a level that was only 0.1 to 10% that achieved by ES114. In addition, class III strains were defective in two capabilities, hemagglutination and luminescence, that have been previously described as colonization factors in
V. fischeri
. Class II and III mutants also share a mucoid colony morphology; however, class II mutants can colonize
E. scolopes
to a level that was 40% of that achieved by ES114. Thus, the mucoid phenotype alone does not contribute to the greater defect exhibited by class III strains. When squid were exposed to ES114 and any one of the HS mutant strains as a coinoculation, the parent strain dominated the resulting symbiotic light-organ population. To further investigate the colonization defects of the HS strains, we used confocal laser-scanning microscopy to visualize
V. fischeri
cells in their initial interaction with
E. scolopes
tissue. Compared to ES114, HS strains from all three classes were delayed in two behaviors involved in colonization: (i) aggregation on host-derived mucus structures and (ii) migration to the crypts. These results suggest that, while motility is required to initiate colonization, the presence of multiple flagella may actually interfere with normal aggregation and attachment behavior. Furthermore, the pleiotropic nature of class III HS strains provides evidence that motility is coregulated with other symbiotic determinants in
V. fischeri
.
Molecular Identification of Family 38 α-Mannosidase ofBacillussp. Strain GL1, Responsible for Complete Depolymerization of Xanthan
Authors: Nankai Hirokazu , Hashimoto Wataru , Murata Kousaku
Published: July 2002

When cells ofBacillussp. strain GL1 were grown in a medium containing xanthan as a carbon source, α-mannosidase exhibiting activity towardp-nitrophenyl-α-d-mannopyranoside (pNP-α-d-Man) was produced intracellularly. The 350-kDa α-mannosidase purified from a cell extract of the bacterium was a trimer comprising three identical subunits, each with a molecular mass of 110 kDa. The enzyme hydrolyzedpNP-α-d-Man (Km= 0.49 mM) andd-mannosyl-(α-1,3)-d-glucose most efficiently at pH 7.5 to 9.0, indicating that the enzyme catalyzes the last step of the xanthan depolymerization pathway ofBacillussp. strain GL1. The gene for α-mannosidase cloned most by using N-terminal amino acid sequence information contained an open reading frame (3,144 bp) capable of coding for a polypeptide with a molecular weight of 119,239. The deduced amino acid sequence showed homology with the amino acid sequences of α-mannosidases belonging to glycoside hydrolase family 38.
Parasporin, a Human Leukemic Cell-Recognizing Parasporal Protein of
Bacillus thuringiensis
Authors: Mizuki Eiichi , Park Yu Shin , Saitoh Hiroyuki , Yamashita Satoko , Akao Tetsuyuki , Higuchi Kazuhiko , Ohba Michio
Published: July 2002

An unusual property, human leukemic cell-recognizing activity, associated with parasporal inclusions of a noninsecticidal
Bacillus thuringiensis
soil isolate was investigated, and a protein (named parasporin in this study) responsible for the activity was cloned. The parasporin, encoded by a gene 2,169 bp long, was a polypeptide of 723 amino acid residues with a predicted molecular weight of 81,045. The sequence of parasporin contained the five conserved blocks commonly found in
B. thuringiensis
Cry proteins; however, only very low homologies (<25%) between parasporin and the existing classes of Cry and Cyt proteins were detected. Parasporin exhibited cytocidal activity only when degraded by proteases into smaller molecules of 40 to 60 kDa. Trypsin and proteinase K activated parasporin, while chymotrypsin did not. The activated parasporin showed strong cytocidal activity against human leukemic T cells (MOLT-4) and human uterus cervix cancer cells (HeLa) but not against normal T cells.
Use of Two-Dimensional Gel Electrophoresis To Measure Changes in Synovial Fluid Proteins from Patients with Rheumatoid Arthritis Treated with Antibody to CD4
Authors: Smith Marjorie A. , Bains Satbinder K. , Betts Joanna C. , Choy Ernest H. S. , Zanders Edward D.
Published: July 2002

Synovial fluid proteins from microliter volumes of synovial fluid were resolved by two-dimensional polyacrylamide gel electrophoresis and detected by silver staining to investigate the feasibility of using two-dimensional (2D) electrophoresis in the clinical research setting and provide global disease information of disease progression. Several hundred proteins could be resolved as spots, many of which displayed the characteristic pattern of plasma-derived glycoproteins. The lowest level of detection was approximately 0.2 ng from a total of 50 μg of protein loaded. Most of the proteins could be identified on the basis of pI and molecular weight when compared with plasma protein maps on the World Wide Web. Unknown proteins were characterized by mass spectrometry of tryptic digests and by comparison with peptide databases. Synovial fluids from patients with rheumatoid arthritis were analyzed using this technique. Each subject received a fixed dose of antibody to CD4 as part of a phase II clinical trial to determine the efficacy of this immunosuppressive treatment in modifying disease activity. Synovial fluid was removed at day 0, followed by administration of antibody. Subsequent removal of synovial fluid and additional administration of antibody were carried out at different times thereafter. Changes in levels of acute-phase proteins were quantified by densitometry of silver-stained 2D polyacrylamide gels. Other parameters of disease progression such as serum C-reactive protein and physician's global assessment of clinical condition were used for comparison. In this way, changes in acute-phase proteins towards normal levels, as measured by 2D polyacrylamide gel electrophoresis, could be correlated with clinical improvement and conventional clinical chemistry measurements. Thus, the system can be used for quantitative analysis of protein expression in sites of autoimmune disease activity such as the synovial fluid of rheumatoid arthritis patients.
Duration of Sample Storage Dramatically Alters Expression of the Human Immunodeficiency Virus Coreceptors CXCR4 and CCR5
Authors: Shalekoff Sharon , Tiemessen Caroline T.
Published: July 2002

Expression of the chemokine receptors CXCR4 and CCR5 was monitored using EDTA-anticoagulated whole blood held for different time periods prior to fluorescent-antibody staining. When left overnight CXCR4 expression on leukocytes was substantially increased, whereas CCR5 expression was reduced. The results were similar when heparin and acid-citrate-dextrose were used as anticoagulants.
New Assay for Measuring Cell Surface Hydrophobicities of
Candida dubliniensis
and
Candida albicans
Authors: Jabra-Rizk M. A. , Falkler W. A. , Merz W. G. , Meiller T. F.
Published: July 2002

Hydrophobic interactions, based on cell surface hydrophobicity (CSH), are among the many and varied mechanisms of adherence deployed by the pathogenic yeast
Candida albicans
. Recently it was shown that, unlike
C. albicans
,
C. dubliniensis
is a species that exhibits an outer fibrillar layer consistent with constant CSH. Previously,
C. dubliniensis
grown at 25 or 37°C was shown to coaggregate with the oral anaerobic bacterium
Fusobacterium nucleatum. C. albicans
, however, demonstrated similar coaggregation only when hydrophobic or grown at 25°C. This observation implied that coaggregation of
Candida
cells with
F. nucleatum
is associated with a hydrophobic yeast cell surface. To test this hypothesis, 42
C. albicans
and 40
C. dubliniensis
clinical isolates, including a
C. albicans
hydrophobic variant, were grown at 25 and 37°C and tested with the established hydrophobicity microsphere assay, which determines CSH levels based on the number of microspheres attached to the yeast cells. The coaggregation assay was performed in parallel experiments. All
C. dubliniensis
isolates grown at either temperature, hydrophobic 25°C-grown
C. albicans
isolates, and the
C. albicans
hydrophobic variant, unlike the 37°C-hydrophilic
C. albicans
isolates, exhibited hydrophobic CSH levels with the microsphere assay and simultaneously showed maximum, 4+, coaggregation with
F. nucleatum
. The parallel results obtained for
C. dubliniensis
using both assays support the use of the CoAg assay both as a rapid assay to determine CSH and to differentiate between
C. dubliniensis
and
C. albicans
.
Clinical and Serological Follow-Up of Patients with Human Granulocytic Ehrlichiosis in Slovenia
Authors: Lotrič-Furlan Stanka , Avsic-Zupanc T. , Petrovec M. , Nicholson W. L. , Sumner J. W. , Childs J. E. , Strle F.
Published: July 2002

An evaluation of the clinical outcome and the duration of the antibody response of patients with human granulocytic ehrlichiosis (HGE) was undertaken in Slovenia. Adult patients with a febrile illness occurring within 6 weeks of a tick bite were classified as having probable or confirmed HGE based on the outcome of serological or PCR testing. Thirty patients (median age, 44 years) were enrolled, and clinical evaluations and serum collection were undertaken at initial presentation and at 14 days, 6 to 8 weeks, and 3 to 4, 6, 12, 18, and 24 months. An indirect immunofluorescence assay (IFA) was performed, and reciprocal titers of ≥128 were interpreted as positive. Patients presented a median of 4 days after the onset of fever and were febrile for a median of 7.5 days; four (13.3%) received doxycycline. Seroconversion was observed in 3 of 30 (10.0%) patients, and 25 (83.3%) showed >4-fold change in antibody titer. PCR results were positive in 2 of 3 (66.7%) seronegative patients but in none of 27 seropositive patients at the first presentation. IFA antibody titers of ≥128 were found in 14 of 29 (48.3%), 17 of 30 (56.7%), 13 of 30 (43.4%), and 12 of 30 (40.0%) patients 6, 12, 18, and 24 months after presentation, respectively. Patients reporting additional tick bites during the study had significantly higher antibody titers at most time points during follow-up. No long-term clinical consequences were found during follow-up.
Diagnosis of Babesiosis Using an Immunoblot Serologic Test
Authors: Ryan Raymond , Krause Peter J. , Radolf Justin , Freeman Kathy , Spielman Andrew , Lenz Ronald , Levin Andrew
Published: July 2002

Although the current indirect immunofluorescent assay (IFA) diagnostic antibody test for human babesiosis is sensitive and specific, an immunoblot antibody test may be easier to standardize and to perform. Our objective, therefore, was to determine the efficacy of and develop interpretive criteria for an immunoblot antibody test for diagnosing acute human babesiosis using a
Babesia microti
whole-cell lysate as the antigen. We compared the reactivity of sera to a
B. microti
immunoblot assay in 24 human subjects experiencing symptoms and expressing laboratory evidence of babesiosis, 28 subjects who experienced Lyme disease, 12 subjects who experienced human granulocytic ehrlichiosis, and 51 subjects who reported no history of any of these diseases and whose sera did not react against
B. microti
antigen in an IFA test. Immunoblot strips were impregnated with proteins derived from the GI strain of
B. microti
that had been electrophoresed in an acrylamide sodium dodecyl sulfate gel, followed by electroblotting onto nitrocellulose membranes. The sera of all subjects who experienced babesiosis reacted against the
B. microti
antigen in the IFA and against at least one of nine immunoblot protein bands specific to
B. microti
. In contrast, none of the sera from people who appeared not to have experienced this infection reacted against the
B. microti
antigen in the IFA (compared to 4% in the immunoblot assay). When two reactive bands were considered as definitive, immunoblot test sensitivity was 96%, while specificity was 99% and predictive positivity and predictive negativity were 96 and 99%, respectively. Our
B. microti
immunoblot procedure shows promise as a sensitive, specific, and reproducible assay for routine clinical diagnosis of acute babesiosis.
Circulating Inflammatory Mediators in Patients with Fever: Predicting Bloodstream Infection
Authors: Groeneveld A. B. Johan , Bossink Ailko W. J. , van Mierlo Gerard J. , Hack C. Erik
Published: July 2002

The systemic host response to microbial infection involves clinical signs and symptoms of infection, including fever and elevated white blood cell (WBC) counts. In addition, inflammatory mediators are released, including activated complement product C3a, interleukin 6 (IL-6), and the acute-phase reactant secretory phospholipase A
2
(sPLA
2
). To compare the value of the latter with the former in predicting (the degree of) microbial infection at the bedside, we determined clinical variables and took blood samples daily for 3 consecutive days in 300 patients with a new fever (>38.0°C rectally or >38.3°C axillary). Microbiological culture results for 7 days after inclusion were collected. Patients were divided into clinical and microbial categories: those without and with a clinical focus of infection and those with negative cultures, with positive local cultures or specific stains for fungal (
n
= 13) or tuberculous infections (
n
= 1), and with positive blood cultures, including one patient with malaria parasitemia. The area under the curve (AUC) of the receiver operating characteristic (ROC) for prediction of positive cultures was 0.60 (
P
< 0.005) for peak temperature and 0.59 (
P
< 0.01) for peak WBC count, 0.60 (
P
< 0.005) for peak C3a, 0.63 (
P
< 0.001) for peak IL-6, and 0.61 (
P
< 0.001) for peak sPLA
2
. The AUC under the ROC curve for prediction of positive blood cultures was 0.68 (
P
< 0.001) for peak temperature and 0.56 for peak WBC count (
P
< 0.05). The AUC for peak C3a was 0.69, that for peak IL-6 was 0.70, and that for sPLA
2
was 0.67 (for all,
P
< 0.001). The degree of microbial invasion is thus a major determinant of the clinical and inflammatory host response in patients with fever. Moreover, circulating inflammatory mediators such as C3a and IL-6 may help to predict positive blood cultures, together with clinical signs and symptoms of the host response to microbial infection, even before culture results are available. This may help in the designing of entry criteria for therapeutic intervention studies.
Isolation and Characterization of Two European Strains ofEhrlichia phagocytophilaof Equine Origin
Authors: Bjöersdorff Anneli , Bagert Bodil , Massung Robert F. , Gusa Asiya , Eliasson Ingvar
Published: July 2002

We report the isolation and partial genetic characterization of two equine strains of granulocyticEhrlichiaof the genogroupEhrlichia phagocytophila. Frozen whole-blood samples from two Swedish horses with laboratory-verified granulocytic ehrlichiosis were inoculated into HL-60 cell cultures. GranulocyticEhrlichiawas isolated and propagated from both horses. DNA extracts from the respective strains were amplified by PCR using primers directed towards the 16S rRNA gene, thegroESLheat shock operon gene, and theankgene. The amplified gene fragments were sequenced and compared to known sequences in the GenBank database. With respect to the 16S rRNA gene, thegroESLgene, and theankgene, the DNA sequences of the two equineEhrlichiaisolates were identical to sequences found in isolates from clinical cases of granulocytic ehrlichiosis in humans and domestic animals in Sweden. However, compared to amplified DNA from an AmericanEhrlichiastrain of theE.phagocytophilagenogroup, differences were found in thegroESLgene andankgene sequences.
Current Status of Malaria and Potential for Control
Authors: Phillips R. S.
Published: July 2002

Malaria remains one of the world's worst health problems with 1.5 to 2.7 million deaths annually; these deaths are primarily among children under 5 years of age and pregnant women in sub-Saharan Africa. Of significance, more people are dying from malaria today than 30 years ago. This review considers the factors which have contributed to this gloomy picture, including those which relate to the vector, the female anopheline mosquito; to human activity such as creating new mosquito breeding sites, the impact of increased numbers of people, and how their migratory behavior can increase the incidence and spread of malaria; and the problems of drug resistance by the parasites to almost all currently available antimalarial drugs. In a selective manner, this review describes what is being done to ameliorate this situation both in terms of applying existing methods in a useful or even crucial role in control and prevention and in terms of new additions to the antimalarial armory that are being developed. Topics covered include biological control of mosquitoes, the use of insecticide-impregnated bed nets, transgenic mosquitoes manipulated for resistance to malaria parasites, old and new antimalarial drugs, drug resistance and how best to maintain the useful life of antimalarials, immunity to malaria and the search for antimalarial vaccines, and the malaria genome project and the potential benefits to accrue from it.
Bacterial Pathogens Induce Abscess Formation by CD4+T-Cell Activation via the CD28–B7-2 Costimulatory Pathway
Authors: Tzianabos Arthur O. , Chandraker Anil , Kalka-Moll Wiltrud , Stingele Francesca , Dong Victor M. , Finberg Robert W. , Peach Robert , Sayegh Mohamed H.
Published: July 2002

Abscesses are a classic host response to infection by many pathogenic bacteria. The immunopathogenesis of this tissue response to infection has not been fully elucidated. Previous studies have suggested that T cells are involved in the pathologic process, but the role of these cells remains unclear. To delineate the mechanism by which T cells mediate abscess formation associated with intra-abdominal sepsis, the role of T-cell activation and the contribution of antigen-presenting cells via CD28-B7 costimulation were investigated. T cells activated in vitro by zwitterionic bacterial polysaccharides (Zps) known to induce abscess formation required CD28-B7 costimulation and, when adoptively transferred to the peritoneal cavity of naı̈ve rats, promoted abscess formation. Blockade of T-cell activation via the CD28-B7 pathway in animals with CTLA4Ig prevented abscess formation following challenge with different bacterial pathogens, includingStaphylococcus aureus,Bacteroides fragilis, and a combination ofEnterococcus faeciumandBacteroides distasonis. In contrast, these animals had an increased abscess rate following in vivo T-cell activation via CD28 signaling. Abscess formation in vivo and T-cell activation in vitro required costimulation by B7-2 but not B7-1. These results demonstrate that abscess formation by pathogenic bacteria is under the control of a common effector mechanism that requires T-cell activation via the CD28–B7-2 pathway.
ThetprKGene Is Heterogeneous amongTreponema pallidumStrains and Has Multiple Alleles
Authors: Centurion-Lara Arturo , Godornes Charmie , Castro Christa , Van Voorhis Wesley C. , Lukehart Sheila A.
Published: July 2002

We have previously shown that the TprK antigen ofT. pallidum, Nichols strain, is predominantly expressed in treponemes obtained 10 days after infection and that the hydrophilic domain of TprK is a target of opsonic antibodies and confers significant protection against homologous challenge. TheT. pallidumgenome sequence reported the presence of a single copy of thetprKgene in the Nichols strain. In the present study we demonstrate size heterogeneity in the central portions of the TprK hydrophilic domains of 14 treponemal isolates. Sequence analysis of the central domains and the complete open reading frames (ORFs) of thetprKgenes confirms this heterogeneity. Further, multipletprKsequences were found in the Nichols-definedtprKlocus in three isolates (Sea 81-4, Bal 7, and Bal 73-1). In contrast, only a singletprKsequence could be identified in this locus in the Nichols strain. Alignment of the DNA and deduced amino acid sequences of the wholetprKORFs shows the presence of seven discrete variable domains flanked by highly conserved regions. We hypothesize that these heterogeneous regions may be involved in antigenic heterogeneity and, in particular, evasion of the immune response. The presence of differenttprKalleles in thetprKlocus strongly suggests the existence of genetically different subpopulations within treponemal isolates.
Identification of a
Treponema denticola
OppA Homologue That Binds Host Proteins Present in the Subgingival Environment
Authors: Fenno J. Christopher , Tamura Muneaki , Hannam Pauline M. , Wong Grace W. K. , Chan Roger A. , McBride Barry C.
Published: July 2002

Proteins secreted or exported by
Treponema denticola
have been implicated as mediators of specific interactions between the spirochete and subgingival tissues in periodontal diseases. However, limited information is available on the ability of this peptidolytic organism to bind or transport soluble peptides present in the subgingival environment. A prominent 70-kDa protein was isolated from surface extracts of
T. denticola
ATCC 35405. A clone expressing a portion of the protein was identified in an
Escherichia coli
expression library of
T. denticola
DNA. DNA sequence analysis showed that the cloned gene encoded a peptide homologous to OppA, the solute binding protein of an ATP-binding cassette-type peptide transporter involved in peptide uptake and environmental signaling in a wide range of bacteria. Genes encoding OppB, -C, -D, and -F were identified directly downstream of
oppA
in
T. denticola
. OppA was present in representative strains of
T. denticola
and in
Treponema vincentii
but was not detected in
Treponema pectinovorum
or
Treponema socranskii
. Immunogold electron microscopy suggested that OppA was accessible to proteins at the surface of the spirochete. Native OppA bound soluble plasminogen and fibronectin but did not bind to immobilized substrates or epithelial cells. A
T. denticola oppA
mutant bound reduced amounts of soluble plasminogen, and plasminogen binding to the parent strain was inhibited by the lysine analog ɛ-aminocaproic acid. Binding of soluble host proteins by OppA may be important both for spirochete-host interactions in the subgingival environment and for uptake of peptide nutrients.
Reduced Virulence of
HWP1
-Deficient Mutants of
Candida albicans
and Their Interactions with Host Cells
Authors: Tsuchimori Noboru , Sharkey Laura L. , Fonzi William A. , French Samuel W. , Edwards John E. , Filler Scott G.
Published: July 2002

The
Candida albicans
gene
HWP1
encodes a surface protein that is required for normal hyphal development in vitro. We used mutants lacking one or both alleles of
HWP1
to investigate the role of this gene in virulence. Mice infected intravenously with the homozygous
hwp1
null mutant, CAL3, survived a median of >14 days, whereas mice infected with a control strain containing two functional alleles of
HWP1
survived only 3.5 days. After 1 day of infection, all strains produced similar levels of infection in the kidneys, spleen, and blood. However, after 2 and 3 days, there was a significant decrease in the number of organisms in the kidneys of the mice infected with CAL3. This finding suggests that the
hwp1
homozygous null mutant is normal in its ability to initiate infection but deficient in its capacity to maintain infection. CAL3 also germinated minimally in the kidneys. The ability of the heterozygous null mutant to germinate and cause mortality in mice was intermediate to CAL3, suggesting a gene dosage effect. To investigate potential mechanisms for the diminished virulence of CAL3, we examined its interactions with endothelial cells and neutrophils in vitro. CAL3 caused less endothelial cell injury than the heterozygous
hwp1
mutant. We conclude that the
HWP1
gene product is important for both in vivo hyphal development and pathogenicity of
C. albicans
. Also, the ability to form filaments may be critical for candidal virulence by enabling the fungus to induce cellular injury and maintain a deep-seated infection.
Host Resistance and Immune Deviation in Pigeon CytochromecT-Cell Receptor Transgenic Mice Infected withToxoplasma gondii
Authors: Collazo Carmen M. , Miller Carla , Yap George , Hieny Sara , Caspar Patricia , Schwartz Ronald H. , Sher Alan
Published: July 2002

Resistance toToxoplasma gondiihas been shown to be mediated by gamma interferon (IFN-γ) produced by NK, CD4+, and CD8+T cells. While studies of SCID mice have implicated NK cells as the source of the cytokine in acute infection, several lines of evidence suggest that IFN-γ production by CD4+T lymphocytes also plays an important role in controlling early parasite growth. To evaluate whether this function is due to nonspecific as opposed to T-cell receptor (TCR)-dependent stimulation by the parasite, we have examined the resistance toT. gondiiinfection of pigeon cytochromectransgenic (PCC-Tg) Rag-2−/−mice in which all CD4+T lymphocytes are unreactive with the protozoan. When inoculated with the ME49 strain, PCC-Tg animals exhibited only temporary control of acute infection and succumbed by day 17. Intracellular cytokine staining by flow cytometry revealed that, in contrast to infected nontransgenic controls, infected PCC-Tg animals failed to develop IFN-γ-producing CD4+T cells. Moreover, the CD4+lymphocytes from these mice showed no evidence of activation as judged by lack of upregulated expression of CD44 or CD69. Nevertheless, when acutely infected transgenic mice were primed by PCC injection, the lymphokine responses measured after in vitro antigen restimulation displayed a strong Th1 bias which was shown to be dependent on endogenous interleukin 12 (IL-12). The above findings argue that, whileT. gondii-induced IL-12 cannot trigger IFN-γ production by CD4+T cells in the absence of TCR ligation, the pathogen is able to nonspecifically promote Th1 responses against nonparasite antigens, an effect that may explain the immunostimulatory properties ofT. gondiiinfection.
Blockade of Costimulation Prevents Infection-Induced Immunopathology in Interleukin-10-Deficient Mice
Authors: Villegas Eric N. , Wille Ulrike , Craig Linden , Linsley Peter S. , Rennick Donna M. , Peach Robert , Hunter Christopher A.
Published: July 2002

Interleukin-10 (IL-10) is associated with inhibition of cell-mediated immunity and downregulation of the expression of costimulatory molecules required for T-cell activation. When IL-10-deficient (IL-10KO) mice are infected withToxoplasma gondii, they succumb to a T-cell-mediated shock-like reaction characterized by the overproduction of IL-12 and gamma interferon (IFN-γ) associated with widespread necrosis of the liver. Since costimulation is critical for T-cell activation, we investigated the role of the CD28-B7 and CD40-CD40 ligand (CD40L) interactions in this infection-induced immunopathology. Our studies show that infection of mice withT. gondiiresulted in increased expression of B7 and CD40 that was similar in wild-type and IL-10KO mice. In vivo blockade of the CD28-B7 or CD40-CD40L interactions following infection of IL-10KO mice withT. gondiidid not affect serum levels of IFN-γ or IL-12, nor did it prevent death in these mice. However, when both pathways were blocked, the IL-10KO mice survived the acute phase of infection and had reduced serum levels of IFN-γ and alanine transaminase as well as decreased expression of inducible nitric oxide synthase in the liver and spleen. Analysis of parasite-specific recall responses from infected IL-10KO mice revealed that blockade of the CD40-CD40L interaction had minimal effects on cytokine production, whereas blockade of the CD28-B7 interaction resulted in decreased production of IFN-γ but not IL-12. Further reduction of IFN-γ production was observed when both costimulatory pathways were blocked. Together, these results demonstrate that the CD28-B7 and CD40-CD40L interactions are involved in the development of infection-induced immunopathology in the absence of IL-10.
Impairment of Endotoxin-Induced Macrophage Inflammatory Protein 2 Gene Expression in Alveolar Macrophages in Streptozotocin-Induced Diabetes in Mice
Authors: Amano Hideaki , Yamamoto Hidefumi , Senba Masachika , Oishi Kazunori , Suzuki Shoichi , Fukushima Kenichi , Mukaida Naofumi , Matsushima Kouji , Eguchi Katsumi , Nagatake Tsuyoshi
Published: July 2002

To elucidate the mechanism of the high incidence of lower respiratory tract infections in patients with diabetes mellitus, we investigated the kinetics of production of macrophage inflammatory protein 2 (MIP-2), an important mediator of lung neutrophil recruitment, using mice with streptozotocin-induced diabetes. Intratracheal challenge with 1 mg of lipopolysaccharide (LPS), an endotoxin, per kg of body weight resulted in a time-dependent increase in the levels of MIP-2 protein in bronchoalveolar lavage (BAL) fluid, with the peak concentration (49.4 ± 13 ng/ml) occurring at 3 h and significant neutrophil accumulation becoming apparent by 3 h in normal mice. In diabetic mice, the peak level of MIP-2 protein in BAL fluid did not occur until 6 h and was reduced to 21.9 ± 10 ng/ml. Immunohistochemical studies using anti-MIP-2 antibody confirmed that the main cellular source of MIP-2 in the lung after LPS challenge was alveolar macrophages (AMs) in normal mice. The lungs in diabetic mice, however, showed no AMs staining for MIP-2 within 3 h after LPS challenge. PCR analysis using whole-lung RNA showed a time-dependent increase in MIP-2 mRNA levels after LPS instillation. The level of MIP-2 mRNA in diabetic mice was markedly decreased compared to that in normal mice. Our results indicate that impairment of MIP-2 mRNA expression in the AMs in diabetic mice resulted in delayed neutrophil recruitment in the lungs, and this may explain the development and progression of pulmonary infection in diabetes mellitus.
Afa/Dr Diffusely Adhering
Escherichia coli
C1845 Infection Promotes Selective Injuries in the Junctional Domain of Polarized Human Intestinal Caco-2/TC7 Cells
Authors: Peiffer Isabelle , Blanc-Potard Anne-Béatrice , Bernet-Camard Marie-Françoise , Guignot Julie , Barbat Alain , Servin Alain L.
Published: July 2002

The Afa/Dr diffusely adhering
Escherichia coli
(DAEC) C1845 strain harboring the F1845 fimbrial adhesin interacts with the brush border-associated CD55 molecule and promotes elongation of brush border microvilli resulting from rearrangement of the F-actin network. This phenomenon involves the activation of a cascade of signaling coupled to the glycosylphosphatidylinositol-anchored receptor of the F1845 adhesin. We provide evidence that infection of the polarized human intestinal cell line Caco-2/TC7 by strain C1845 is followed by an increase in the paracellular permeability for [
3
H]mannitol without a decrease of the transepithelial resistance of the monolayers. Alterations in the distribution of tight-junction (TJ)-associated occludin and ZO-1 protein are observed, whereas the distribution of the zonula adherens-associated E-cadherin is not affected. Using the recombinant
E. coli
strains HB101(pSSS1) and -(pSSS1C) expressing the F1845 fimbrial adhesin, we demonstrate that the adhesin-CD55 interaction is not sufficient for the induction of structural and functional TJ lesions. Moreover, using the actin filament-stabilizing agent Jasplakinolide, we demonstrate that the C1845-induced functional alterations in TJs are independent of the C1845-induced apical cytoskeleton rearrangements. The results indicated that pathogenic factor(s) other than F1845 adhesin may be operant in Afa/Dr DAEC C1845.
Role of Hyphal Formation in Interactions of
Candida albicans
with Endothelial Cells
Authors: Phan Quynh T. , Belanger Paul H. , Filler Scott G.
Published: July 2002

The ability to change from yeast to hyphal morphology is a major virulence determinant of
Candida albicans
. Mutants with defined defects in filamentation regulatory pathways have reduced virulence in mice. However, is it poorly understood why hyphal formation is critical for
C. albicans
to cause hematogenously disseminated infections. We used recently constructed mutants to examine the role of hyphal formation in the interactions of
C. albicans
with endothelial cells in vitro. These interactions included the ability of the mutants to invade and injure endothelial cells. Because the formation of hyphae may influence the host inflammatory response to
C. albicans
, we also investigated the capacity of these mutants to stimulate endothelial cells to express E-selectin and intercellular adhesion molecule 1. We infected endothelial cells with
C. albicans
strains containing homozygous null mutations in the following filamentation regulatory genes:
CLA4
,
CPH1
,
EFG1
, and
TUP1
. Whereas the wild-type strain formed true hyphae on endothelial cells, we found that neither the Δ
efg1
nor the Δ
cph1 Δefg1
double mutant germinated. The Δ
tup1
mutant formed only pseudohyphae. We also found that the Δ
efg1
, Δ
cph1 Δefg1
, and Δ
tup1
mutants had significantly reduced capacities to invade and injure endothelial cells. Therefore, Efg1p and Tup1p contribute to virulence by regulating hyphal formation and the factors that enable
C. albicans
to invade and injure endothelial cells. With the exception of the
Δcph1 Δefg1
mutant, all other mutants stimulated endothelial cells to express at least one of the leukocyte adhesion molecules. Therefore, the combined activities of Cph1p and Efg1p are required for
C. albicans
to stimulate a proinflammatory response in endothelial cells.