bluuebunny/crossref_metadata_2025 huggingface dataset:
Randomized Controlled Trial of Sequential Intravenous (i.v.) and Oral Moxifloxacin Compared with Sequential i.v. and Oral Co-Amoxiclav with or without Clarithromycin in Patients with Community-Acquired Pneumonia Requiring Initial Parenteral Treatment
Authors: Finch R. , Schürmann D. , Collins O. , Kubin R. , McGivern J. , Bobbaers H. , Izquierdo J. L. , Nikolaides P. , Ogundare F. , Raz R. , Zuck P. , Hoeffken G.
Published: July 2002

The objective of the present trial was to compare the efficacy, safety, and tolerability of moxifloxacin (400 mg) given intravenously (i.v.) once daily followed by oral moxifloxacin (400 mg) for 7 to 14 days with the efficacy, safety, and tolerability of co-amoxiclav (1.2 g) administered by i.v. infusion three times a day followed by oral co-amoxiclav (625 mg) three times a day, with or without clarithromycin (500 mg) twice daily (i.v. or orally), for 7 to 14 days in adult patients with community-acquired pneumonia requiring initial parenteral therapy. A total of 628 patients were enrolled and assessed by evaluation of their clinical and bacteriological responses 5 to 7 days and 21 to 28 days after administration of the last dose of study medication. Although the trial was designed, on the basis of predefined outcomes, to demonstrate the equivalence of the two regimens, the results showed statistically significant higher clinical success rates (for moxifloxacin, 93.4%, and for comparator regimen, 85.4%; difference [Δ], 8.05%; 95% confidence interval [CI], 2.91 to 13.19%;
P
= 0.004) and bacteriological success rates (for moxifloxacin, 93.7%, and for comparator regimen, 81.7%; Δ, 12.06%; 95% CI, 1.21 to 22.91%) for patients treated with moxifloxacin. This superiority was seen irrespective of the severity of the pneumonia and whether or not the combination therapy included a macrolide. The time to resolution of fever was also statistically significantly faster for patients who received moxifloxacin (median time, 2 versus 3 days), and the duration of hospital admission was approximately 1 day less for patients who received moxifloxacin. The treatment was converted to oral therapy immediately after the initial mandatory 3-day period of i.v. administration for a larger proportion of patients in the moxifloxacin group than patients in the comparator group (151 [50.2%] versus 57 [17.8%] patients). There were fewer deaths (9 [3.0%] versus 17 [5.3%]) and fewer serious adverse events (38 [12.6%] versus 53 [16.5%]) in the moxifloxacin group than in the comparator group. The rates of drug-related adverse events were comparable in both groups (38.9% in each treatment group). The overall incidence of laboratory abnormalities was similar in both groups. Thus, it is concluded that monotherapy with moxifloxacin is superior to that with a standard combination regimen of a β-lactam and a β-lactamase inhibitor, co-amoxiclav, with or without a macrolide, clarithromycin, in the treatment of patients with community-acquired pneumonia admitted to a hospital.
mRNA Detection by Reverse Transcription-PCR for Monitoring Viability over Time in an
Enterococcus faecalis
Viable but Nonculturable Population Maintained in a Laboratory Microcosm
Authors: del Mar Lleò Maria , Pierobon Sabrina , Tafi Maria Carla , Signoretto Caterina , Canepari Pietro
Published: July 2002

The viable but nonculturable (VBNC) state is a survival strategy adopted by bacteria when they are exposed to hostile environmental conditions. It has been shown that VBNC forms of bacteria are no longer capable of growing on conventional bacteriological media but conserve pathogenic factors and/or genes. It is thus necessary to develop methods capable of detecting nonculturable bacteria and of establishing their viability when the microbiological quality of environments is monitored. In this study we demonstrated that a gene was expressed during the VBNC state in a low-nutrient-concentration microcosm through detection of
Enterococcus faecalis pbp5
mRNA by reverse transcription-PCR over a 3-month period. The presence of mRNA correlated with metabolic activity and resuscitation capability, indicating the viability of the VBNC cells.
Molecular Characterization and Identification of Biocontrol Isolates of
Trichoderma
spp
Authors: Hermosa M. R. , Grondona I. , Iturriaga E. A. , Diaz-Minguez J. M. , Castro C. , Monte E. , Garcia-Acha I.
Published: July 2002

The most common biological control agents (BCAs) of the genus
Trichoderma
have been reported to be strains of
Trichoderma virens
,
T. harzianum
, and
T. viride
. Since
Trichoderma
BCAs use different mechanisms of biocontrol, it is very important to explore the synergistic effects expressed by different genotypes for their practical use in agriculture. Characterization of 16 biocontrol strains, previously identified as “
Trichoderma harzianum
” Rifai and one biocontrol strain recognized as
T. viride
, was carried out using several molecular techniques. A certain degree of polymorphism was detected in hybridizations using a probe of mitochondrial DNA. Sequencing of internal transcribed spacers 1 and 2 (ITS1 and ITS2) revealed three different ITS lengths and four different sequence types. Phylogenetic analysis based on ITS1 sequences, including type strains of different species, clustered the 17 biocontrol strains into four groups:
T. harzianum-T. inhamatum
complex,
T. longibrachiatum
,
T. asperellum
, and
T. atroviride-T. koningii
complex. ITS2 sequences were also useful for locating the biocontrol strains in
T. atroviride
within the complex
T. atroviride-T. koningii
. None of the biocontrol strains studied corresponded to biotypes Th2 or Th4 of
T. harzianum
, which cause mushroom green mold. Correlation between different genotypes and potential biocontrol activity was studied under dual culturing of 17 BCAs in the presence of the phytopathogenic fungi
Phoma betae
,
Rosellinia necatrix
,
Botrytis cinerea
, and
Fusarium oxysporum
f. sp.
dianthi
in three different media.
Reductive Dehalogenation of Trichloroacetic Acid by
Trichlorobacter thiogenes
gen. nov., sp. nov
Authors: De Wever Helene , Cole James R. , Fettig Michael R. , Hogan Deborah A. , Tiedje James M.
Published: July 2002

A bacterium able to grow via reductive dechlorination of trichloroacetate was isolated from anaerobic soil enrichments. The isolate, designated strain K1, is a member of the δ proteobacteria and is related to other known sulfur and ferric iron reducers. In anaerobic mineral media supplemented with acetate and trichloroacetate, its doubling time was 6 h. Alternative electron donor and acceptors were acetoin and sulfur or fumarate, respectively. Trichloroacetate dehalogenation activity was constitutively present, and the dechlorination product was dichloroacetate and chloride. Trichloroacetate conversion seemed to be coupled to a novel sulfur-sulfide redox cycle, which shuttled electrons from acetate oxidation to trichloroacetate reduction. In view of its unique physiological characteristics, the name
Trichlorobacter thiogenes
is suggested for strain K1.
Additional Evidence that Juvenile Oyster Disease Is Caused by a Member of the
Roseobacter
Group and Colonization of Nonaffected Animals by
Stappia stellulata
-Like Strains
Authors: Boettcher Katherine J. , Barber Bruce J. , Singer John T.
Published: July 2002

Juvenile oyster disease (JOD) causes significant annual mortalities of hatchery-produced Eastern oysters,
Crassostrea virginica
, cultured in the Northeast. We have reported that a novel species of the α-proteobacteria
Roseobacter
group (designated CVSP) was numerically dominant in JOD-affected animals sampled during the 1997 epizootic on the Damariscotta River, Maine. In this study we report the isolation of CVSP bacteria from JOD-affected oysters during three separate epizootics in 1998. These bacteria were not detected in nonaffected oysters at the enzootic site, nor in animals raised at a JOD-free site. Animals raised at the JOD enzootic site that were unaffected by JOD were stably and persistently colonized by
Stappia stellulata
-like strains. These isolates (designated M1) inhibited the growth of CVSP bacteria in a disk-diffusion assay and thus may have prevented colonization of these animals by CVSP bacteria in situ. Laboratory-maintained
C. virginica
injected with CVSP bacteria experienced statistically significant elevated mortalities compared to controls, and CVSP bacteria were recovered from these animals during the mortality events. Together, these results provide additional evidence that CVSP bacteria are the etiological agent of JOD. Further, there are no other descriptions of specific marine α-proteobacteria that have been successfully cultivated from a defined animal host. Thus, this system presents an opportunity to investigate both bacterial and host factors involved in the establishment of such associations and the role of the invertebrate host in the ecology of these marine α-proteobacteria.
Conversion of Milled Pine Wood by Manganese Peroxidase from
Phlebia radiata
Authors: Hofrichter Martin , Lundell Taina , Hatakka Annele
Published: July 2002

Purified manganese peroxidase (MnP) from the white-rot basidiomycete
Phlebia radiata
was found to convert in vitro milled pine wood (MPW) suspended in an aqueous reaction solution containing Tween 20, Mn
2+
, Mn-chelating organic acid (malonate), and a hydrogen peroxide-generating system (glucose-glucose oxidase). The enzymatic attack resulted in the polymerization of lower-molecular-mass, soluble wood components and in the partial depolymerization of the insoluble bulk of pine wood, as demonstrated by high-performance size exclusion chromatography (HPSEC). The surfactant Tween 80 containing unsaturated fatty acid redsidues promoted the disintegration of bulk MPW. HPSEC showed that the depolymerization yielded preferentially lignocellulose fragments with a predominant molecular mass of ca. 0.5 kDa. MnP from
P. radiata
(MnP3) turned out to be a stable enzyme remaining active for 2 days even at 37°C with vigorous stirring, and 65 and 35% of the activity applied was retained in Tween 20 and Tween 80 reaction mixtures, respectively. In the course of reactions, major part of the Mn-chelator malonate was decomposed (85 to 87%), resulting in an increase of pH from 4.4 to >6.5. An aromatic nonphenolic lignin structure (β-O-4 dimer), which is normally not attacked by MnP, was oxidizible in the presence of pine wood meal. This finding indicates that certain wood components may promote the degradative activities of MnP in a way similar to that promoted by Tween 80, unsaturated fatty acids, or thiols.
Synthesis of Polyhydroxyalkanoate in the Peroxisome of
Saccharomyces cerevisiae
by Using Intermediates of Fatty Acid β-Oxidation
Authors: Poirier Yves , Erard Nadine , Petétot Jean MacDonald-Comber
Published: July 2002

Medium-chain-length polyhydroxyalkanoates (PHAs) are polyesters having properties of biodegradable thermoplastics and elastomers that are naturally produced by a variety of pseudomonads.
Saccharomyces cerevisiae
was transformed with the
Pseudomonas aeruginosa
PHAC1 synthase modified for peroxisome targeting by the addition of the carboxyl 34 amino acids from the
Brassica napus
isocitrate lyase. The PHAC1 gene was put under the control of the promoter of the catalase A gene. PHA synthase expression and PHA accumulation were found in recombinant
S
.
cerevisiae
growing in media containing fatty acids. PHA containing even-chain monomers from 6 to 14 carbons was found in recombinant yeast grown on oleic acid, while odd-chain monomers from 5 to 15 carbons were found in PHA from yeast grown on heptadecenoic acid. The maximum amount of PHA accumulated was 0.45% of the dry weight. Transmission electron microscopy of recombinant yeast grown on oleic acid revealed the presence of numerous PHA inclusions found within membrane-bound organelles. Together, these data show that
S
.
cerevisiae
expressing a peroxisomal PHA synthase produces PHA in the peroxisome using the 3-hydroxyacyl coenzyme A intermediates of the β-oxidation of fatty acids present in the media.
S
.
cerevisiae
can thus be used as a powerful model system to learn how fatty acid metabolism can be modified in order to synthesize high amounts of PHA in eukaryotes, including plants.
Portable System for Microbial Sample Preparation and Oligonucleotide Microarray Analysis
Authors: Bavykin Sergei G. , Akowski James P. , Zakhariev Vladimir M. , Barsky Viktor E. , Perov Alexander N. , Mirzabekov Andrei D.
Published: July 2002

We have developed a three-component system for microbial identification that consists of (i) a universal syringe-operated silica minicolumn for successive DNA and RNA isolation, fractionation, fragmentation, fluorescent labeling, and removal of excess free label and short oligonucleotides; (ii) microarrays of immobilized oligonucleotide probes for 16S rRNA identification; and (iii) a portable battery-powered device for imaging the hybridization of fluorescently labeled RNA fragments with the arrays. The minicolumn combines a guanidine thiocyanate method of nucleic acid isolation with a newly developed hydroxyl radical-based technique for DNA and RNA labeling and fragmentation. DNA and RNA can also be fractionated through differential binding of double- and single-stranded forms of nucleic acids to the silica. The procedure involves sequential washing of the column with different solutions. No vacuum filtration steps, phenol extraction, or centrifugation is required. After hybridization, the overall fluorescence pattern is captured as a digital image or as a Polaroid photo. This three-component system was used to discriminate
Escherichia coli, Bacillus subtilis, Bacillus thuringiensis
, and human HL60 cells. The procedure is rapid: beginning with whole cells, it takes approximately 25 min to obtain labeled DNA and RNA samples and an additional 25 min to hybridize and acquire the microarray image using a stationary image analysis system or the portable imager.
Purification and Characterization of an X-Prolyl-Dipeptidyl Peptidase from
Lactobacillus sakei
Authors: Sanz Yolanda , Toldrá Fidel
Published: July 2002

An X-prolyl-dipeptidyl peptidase has been purified from
Lactobacillus sakei
by ammonium sulfate fractionation and five chromatographic steps, which included hydrophobic interaction, anion-exchange chromatography, and gel filtration chromatography. This procedure resulted in a recovery yield of 7% and an increase in specificity of 737-fold. The enzyme appeared to be a dimer with a subunit molecular mass of approximately 88 kDa. Optimal activity was shown at pH 7.5 and 55°C. The enzyme was inhibited by serine proteinase inhibitors and several divalent cations (Cu
2+
, Hg
2+
, and Zn
2+
). The enzyme almost exclusively hydrolyzed X-Pro from the N terminus of each peptide as well as fluorescent and colorimetric substrates; it also hydrolyzed X-Ala at the N terminus, albeit at lower rates.

K
m

s for Gly-Pro- and Lys-Ala-7-amido-4-methylcoumarin were 29 and 88 μM, respectively; those for Gly-Pro- and Ala-Pro-
p
-nitroanilide were 192 and 50 μM, respectively. Among peptides, β-casomorphin 1-3 was hydrolyzed at the highest rates, while the relative hydrolysis of the other tested peptides was only 1 to 12%. The potential role of the purified enzyme in the proteolytic pathway by catalyzing the hydrolysis of peptide bonds involving proline is discussed.
Best Viral Elution Method Available for Quantification of Enteroviruses in Sludge by Both Cell Culture and Reverse Transcription-PCR
Authors: Monpoeho S. , Maul A. , Mignotte-Cadiergues B. , Schwartzbrod L. , Billaudel S. , Ferré V.
Published: July 2002

The aim of this study was to select one or several virus extraction techniques that enable simultaneous detection of enterovirus genomes and infectious particles in different types of urban sludge. Eight techniques were compared by using 16 different liquid and solid sludge samples. The numbers of infectious enteroviruses in cell cultures were determined by using the most-probable-number method. The enterovirus genome was quantified by a single-tube reverse transcription-PCR using TaqMan technology. The results were statistically analyzed by Friedman's test, a nonparametric test for analysis of randomized block data using only ranks in terms of extraction technique efficiency. Two techniques seemed to yield higher viral titers as determined by simultaneous detection by cell culture and PCR. The first involved a 10% beef extract solution at pH 9 and sonication; the second involved a 0.3 M NaCl–7% beef extract solution at pH 7.5 followed by Freon treatment. In solid sludge, no significant differences were observed among the eight techniques tested. Both of the best techniques can be used for simultaneous detection of infectious enterovirus particles and genomes in any type of urban sludge.
Rapid Evolution of a Sexual Reproduction Gene in Centric Diatoms of the Genus
Thalassiosira
Authors: Armbrust E. Virginia , Galindo H. M.
Published: July 2002

Sexual reproduction is commonly assumed to occur in the vast majority of diatoms due to the intimate association of this process with cell size control. Surprisingly, however, little is known about the impact of sexual events on diatom population dynamics. The
Sig1
gene is strongly upregulated during sexual reproduction in the centric diatom
Thalassiosira weissflogii
and has been hypothesized to encode a protein involved in gamete recognition. In the present study, degenerate PCR primers were designed and used to amplify a portion of
Sig1
from three closely related species in the cosmopolitan genus
Thalassiosira,
Thalassiosira oceanica
,
Thalassiosira guillardii
, and
Thalassiosira pseudonana
. Identification of
Sig1
in these three additional species facilitated development of this gene as a molecular marker for diatom sexual events. Examination of the new sequences indicated that multiple copies of
Sig1
are probably present in the genome. Moreover, compared to the housekeeping gene
β
-tubulin
, the
Sig1
genes of isolates of
T. weissflogii
collected from different regions of the Atlantic and Pacific oceans displayed high levels of divergence. The
Sig1
genes of the four closely related
Thalassiosira
species also displayed high levels of sequence divergence compared to the levels observed with a second gene,
Fcp
, probably explaining why
Sig1
could not be amplified from more distantly related species. The high levels of sequence divergence both within and between species suggest that
Sig1
is rapidly evolving in a manner reminiscent of the manner observed in other genes that encode gamete recognition proteins. A simple model is presented for
Sig1
evolution and the implications of such a rapidly evolving sexual reproduction gene for diatom speciation and population dynamics.
Rapid Confirmation of
Clostridium perfringens
by Using Chromogenic and Fluorogenic Substrates
Authors: Adcock Philip W. , Saint Christopher P.
Published: July 2002

The use of 4-methylumbelliferyl phosphate (MUP) and
ortho
-nitrophenyl-β-
d
-galactopyranoside (ONPG) for the identification of
Clostridium perfringens
was investigated. A liquid assay containing both MUP and ONPG was a highly specific alternative method for
C. perfringens
confirmation, reducing incubation time from 48 to only 4 h. The assay solution is easy to prepare, does not require anaerobic conditions for use, and has an extended shelf life.
Capsule-Transmitted Gut Symbiotic Bacterium of the Japanese Common Plataspid Stinkbug,
Megacopta punctatissima
Authors: Fukatsu Takema , Hosokawa Takahiro
Published: July 2002

The Japanese common plataspid stinkbug,
Megacopta punctatissima
, deposits small brown particles, or symbiont capsules, on the underside of the egg mass for the purpose of transmission of symbiotic bacteria to the offspring. We investigated the microbiological aspects of the bacteria contained in the capsule, such as microbial diversity, phylogenetic placement, localization in vivo, and fitness effects on the host insect. Restriction fragment length polymorphism analysis of 16S ribosomal DNA clones revealed that a single bacterial species dominates the microbiota in the capsule. The bacterium was not detected in the eggs but in the capsules, which unequivocally demonstrated that the bacterium is transmitted to the offspring of the insect orally rather than transovarially, through probing of the capsule content. Molecular phylogenetic analysis showed that the bacterium belongs to the γ-subdivision of the
Proteobacteria
. In adult insects the bacterium was localized in the posterior section of the midgut. Deprivation of the bacterium from the nymphs resulted in retarded development, arrested growth, abnormal body coloration, and other symptoms, suggesting that the bacterium is essential for normal development and growth of the host insect.
Modeling Yeast Spoilage in Cold-Filled Ready-To-Drink Beverages with
Saccharomyces cerevisiae
,
Zygosaccharomyces bailii
, and
Candida lipolytica
Authors: Battey Alyce Stiles , Duffy Siobain , Schaffner Donald W.
Published: July 2002

Mathematical models were developed to predict the probability of yeast spoilage of cold-filled ready-to-drink beverages as a function of beverage formulation. A Box-Behnken experimental design included five variables, each at three levels: pH (2.8, 3.3, and 3.8), titratable acidity (0.20, 0.40, and 0.60%), sugar content (8.0, 12.0, and 16.0 °Brix), sodium benzoate concentration (100, 225, and 350 ppm), and potassium sorbate concentration (100, 225, and 350 ppm). Duplicate samples were inoculated with a yeast cocktail (100 μl/50 ml) consisting of equal proportions of
Saccharomyces cerevisiae
,
Zygosaccharomyces bailii
, and
Candida lipolytica
(∼5.0 × 10
4
CFU/ml each). The inoculated samples were plated on malt extract agar after 0, 1, 2, 4, 6, and 8 weeks. Logistic regression was used to create the predictive models. The pH and sodium benzoate and potassium sorbate concentrations were found to be significant factors controlling the probability of yeast growth. Interaction terms for pH and each preservative were also significant in the predictive model. Neither the titratable acidity nor the sugar content of the model beverages was a significant predictor of yeast growth in the ranges tested.
Degradation of 17β-Estradiol by a Gram-Negative Bacterium Isolated from Activated Sludge in a Sewage Treatment Plant in Tokyo, Japan
Authors: Fujii Katsuhiko , Kikuchi Shintaro , Satomi Masataka , Ushio-Sata Noriko , Morita Naoki
Published: July 2002

A 17β-estradiol (E2)-degrading bacterium was isolated from activated sludge in a sewage treatment plant in Tokyo, Japan. The isolate was suggested to be a new
Novosphingobium
species. Gas chromatography-mass spectrometry and
1
H nuclear magnetic resonance analyses of the metabolites of E2 degradation suggested that no toxic products accumulated in the culture medium.
Structural and Functional Lesions in Brush Border of Human Polarized Intestinal Caco-2/TC7 Cells Infected by Members of the Afa/Dr Diffusely Adhering Family ofEscherichia coli
Authors: Peiffer Isabelle , Guignot Julie , Barbat Alain , Carnoy Christophe , Moseley Steve L. , Nowicki Bogdan J. , Servin Alain L. , Bernet-Camard Marie-Françoise
Published: July 2002

Diffusely adheringEscherichia coli(DAEC) strains expressing F1845 fimbrial adhesin or Dr hemagglutinin belonging to the Afa/Dr family of adhesins infect cultured polarized human intestinal cells through recognition of the brush border-associated decay-accelerating factor (DAF; CD55) as a receptor. The wild-type Afa/Dr DAEC strain C1845 has been shown to induce brush border lesions by an adhesin-dependent mechanism triggering apical F-actin rearrangements. In the present study, we undertook to further characterize cell injuries following the interaction of wild-type Afa/Dr DAEC strains C1845 and IH11128 expressing fimbrial F1845 adhesin and Dr hemagglutinin, respectively, with polarized, fully differentiated Caco-2/TC7 cells. In both cases, bacterium-cell interaction was followed by rearrangement of the major brush border-associated cytoskeletal proteins F-actin, villin, and fimbrin, proteins which play a pivotal role in brush border assembly. In contrast, distribution of G-actin, actin-depolymerizing factor, and tubulin was not modified. UsingdraEmutants, we found that a mutant in which cysteine replaces aspartic acid at position 54 conserved binding capacity but failed to induce F-actin disassembly. Accompanying the cytoskeleton injuries, we found that the distribution of brush border-associated functional proteins sucrase-isomaltase (SI), dipeptidylpeptidase IV (DPPIV), glucose transporter SGLT1, and fructose transporter GLUT5 was dramatically altered. In parallel, SI and DPPIV enzyme activity decreased.
Mutations in the S1 Subunit of Pertussis Toxin That Affect Secretion
Authors: Craig-Mylius Kathleen A. , Stenson Trevor H. , Weiss Alison A.
Published: July 2002

Pertussis toxin is a member of the AB
5
family of toxins and is composed of five subunits (S1 to S5) present in a 1:1:1:2:1 ratio. Secretion is a complex process. Each subunit has a secretion signal that mediates transport to the periplasm, where processing and assembly occur. Secretion of the assembled 105-kDa toxin past the outer membrane is mediated by the nine proteins encoded in the
ptl
operon. Previous studies have shown that S1, the catalytically active A subunit of pertussis toxin, is necessary for efficient secretion, suggesting that a domain on S1 may be required for interaction with the secretion apparatus. Previously, recombinant S1 from four different mutants (serine 54 to glycine, serine 55 to glycine, serine 56 to glycine, and arginine 57 to lysine) was shown to retain catalytic activity. We introduced these mutations into
Bordetella pertussis
and monitored pertussis toxin production and secretion. No pertussis toxin was detected in the serine 54-to-glycine mutant. The other S1 mutants produced periplasmic pertussis toxin, but little pertussis toxin secretion was observed. The arginine 57-to-lysine mutant had the most dramatic secretion defect. It produced wild-type levels of periplasmic pertussis toxin but secreted only 8% as much toxin as the wild-type strain. This phenotype was similar to that observed for strains with mutations in the
ptl
genes, suggesting that this region may have a role in pertussis toxin secretion.
Use of Pertussis Toxin Encoded by
ptx
Genes from
Bordetella bronchiseptica
To Model the Effects of Antigenic Drift of Pertussis Toxin on Antibody Neutralization
Authors: Hausman Sally Z. , Burns Drusilla L.
Published: July 2002

Recently, concern has been voiced about the potential effect that antigenic divergence of circulating strains of
Bordetella pertussis
might have on the efficacy of pertussis vaccines. In order to model antigenic drift of pertussis toxin, a critical component of many pertussis vaccines, and to examine the effects of such drift on antibody neutralization, we engineered a strain of
B. pertussis
to produce a variant pertussis toxin molecule that contains many of the amino acid changes found in the toxin encoded by
Bordetella bronchiseptica ptx
genes. This altered form of the toxin, which is efficiently secreted by
B. pertussis
and which displays significant biological activity, was found to be neutralized by antibodies induced by vaccination as readily as toxin produced by wild-type
B. pertussis
. These findings suggest that significant amino acid changes in the pertussis toxin sequence can occur without drastically altering the ability of antibodies to recognize and neutralize the toxin molecule.
Enhanced Gamma Interferon Production through Activation of Vα14+Natural Killer T Cells by α-Galactosylceramide in Interleukin-18-Deficient Mice with Systemic Cryptococcosis
Authors: Kawakami Kazuyoshi , Kinjo Yuki , Yara Satomi , Uezu Kaori , Koguchi Yoshinobu , Tohyama Masaki , Azuma Masato , Takeda Kiyoshi , Akira Shizuo , Saito Atsushi
Published: July 2002

We showed recently that activation of Vα14+natural killer T cells (NKT cells) by α-galactosylceramide (α-GalCer) resulted in increased gamma interferon (IFN-γ) production and host resistance to intravenous infection withCryptococcus neoformans. In other studies, interleukin-18 (IL-18) activated NKT cells in collaboration with IL-12, suggesting the possible contribution of this cytokine to α-GalCer-induced IFN-γ synthesis. Here we examined the role of IL-18 in α-GalCer-induced Th1 response by using IL-18KO mice with this infection. In these mice, levels of IFN-γ in serum and its synthesis in vitro by spleen cells stimulated with live organisms were not reduced, but rather enhanced, compared to those in wild-type (WT) mice, while such production was completely absent in IL-12KO mice. The enhanced production of IFN-γ correlated with increased IL-12 synthesis but not with reduced production of IL-4, which was rather increased. IFN-γ synthesis in IL-18KO mice was abolished by neutralizing anti-IL-12 antibody and significantly inhibited by neutralization of endogenous IL-4 with a specific monoclonal antibody. In addition, administration of recombinant IL-4 significantly enhanced the production of IFN-γ in WT mice. Finally, the enhanced production of IFN-γ in IL-18KO mice correlated with increased host defense against cryptococcal infection, as indicated by enhancement in α-GalCer-related clearance of microorganisms. Our results indicated that in IL-18KO mice, IFN-γ synthesis was enhanced through overproduction of IL-12 and IL-4 after intravenous infection withC. neoformansand a ligand-specific activation of Vα14+NKT cells.
Functional Characteristics of a Protective Monoclonal Antibody against Serotype A and C Lipooligosaccharides from
Moraxella catarrhalis
Authors: Hu Wei-Gang , Chen Jing , McMichael John C. , Gu Xin-Xing
Published: July 2002

A monoclonal antibody (MAb), designated MAb 8E7 (immunoglobulin G3), specific for
Moraxella catarrhalis
lipooligosaccharide (LOS) was evaluated for its functional activity in vitro and in a mouse model of colonization. Enzyme-linked immunosorbent assay (ELISA) demonstrated that the MAb 8E7 could be prepared to a high titer against LOS of the homologous strain 035E, and that it had bactericidal activity. MAb 8E7 reacted with
M. catarrhalis
serotype A and C LOSs but not serotype B LOS, as measured by ELISA and Western blotting. On the basis of published structures of LOSs, this suggests that the epitope recognized by MAb 8E7 is directed to a common sequence of either α-GlcNAc-(1→2)-β-Glc-(1→ at the branch substituting position 4 of the trisubstituted Glc residue or a terminal tetrasaccharide α-Gal-(1→4)-β-Gal-(1→4)-α-Glc-(1→2)-β-Glc-(1→ at the branch substituting position 6 of the trisubstituted Glc residue. In a whole-cell ELISA, MAb 8E7 reacted with 70% of the 30 wild-type strains and clinical isolates tested. Immuno-electron microscopy demonstrated that MAb 8E7 reacted with a cell surface-exposed epitope of LOS on strain O35E. MAb 8E7 inhibited the adherence of strain O35E to Chang conjunctival epithelial cells by 90%. Passive immunization with MAb 8E7 could significantly enhance the clearance of strain O35E from mouse lungs in an aerosol challenge mouse model. This enhanced bacterial clearance was inhibited when MAb 8E7 was absorbed by
M. catarrhalis
serotype A LOS, indicating that the
M. catarrhalis
LOS-directed antibody may play a major role in the enhancement of
M. catarrhalis
clearance from lungs. These data suggest that MAb 8E7, which recognizes surface-exposed LOS of
M. catarrhalis
, is a protective antibody against
M. catarrhalis
.
Expression of Adhesion Molecules in Synovia of Patients with Treatment-Resistant Lyme Arthritis
Authors: Akin Evren , Aversa John , Steere Allen C.
Published: July 2002

The expression of adhesion molecules in synovium in patients with Lyme arthritis is surely critical in the control ofBorrelia burgdorferiinfection but may also have pathologic consequences. For example, molecular mimicry between a dominant T-cell epitope ofB. burgdorferiouter surface protein A and an adhesion molecule, human lymphocyte function-associated antigen 1 (LFA-1), has been implicated in the pathogenesis of treatment-resistant Lyme arthritis. Using immunohistochemical methods, we examined synovial samples for expression of adhesion molecules in 29 patients with treatment-resistant Lyme arthritis and in 15 patients with rheumatoid arthritis or chronic inflammatory monoarthritis. In Lyme arthritis synovia, endothelial cells showed intense expression of P-selectin and vascular adhesion protein-1 (VAP-1). Expression of LFA-1 was also intense on infiltrating cells, particularly in lymphoid aggregates, and intercellular adhesion molecule-1 (ICAM-1) was markedly expressed on synovial lining and endothelial and infiltrating cells. Moderate expression of vascular cell adhesion molecule-1 (VCAM-1) was seen on synovial lining and endothelial cells, and mild expression of its ligand, very late antigen-4, was apparent in perivascular lymphoid infiltrates. Except for lesser expression of VCAM-1 in Lyme synovia, the levels of expression of these adhesion molecules were similar in the three patient groups. We conclude that certain adhesion molecules, including ICAM-1 and LFA-1, are expressed intensely in the synovia of patients with Lyme arthritis. Upregulation of LFA-1 on lymphocytes in this lesion may be critical in the pathogenesis of treatment-resistant Lyme arthritis.
Critical Role of Cytotoxic T Lymphocytes in Immune Clearance of Rickettsial Infection
Authors: Walker David H. , Olano Juan P. , Feng Hui-Min
Published: July 2002

Cytotoxic T-lymphocyte (CTL) activity developed against the major infected target cells of rickettsial infections, endothelial cells and macrophages. Spleen cells from mice immune toRickettsia conoriiexerted specific major histocompatibility complex (MHC) class I-matched CTL activity againstR. conorii-infected SVEC-10 endothelial cells, with peak activity on day 10. Similarly, spleen cells fromRickettsia australis-immune mice exerted specific CTL activity against anR. australis-infected macrophage-like cell line. Gamma interferon (IFN-γ) gene knockout mice were more than 100-fold more susceptible toR. australisinfection than wild-type C57BL/6 mice. MHC class I gene knockout mice were the most susceptible, more than 50,000-fold more susceptible to a lethal outcome ofR. australisinfection than wild-type C57BL/6 mice. These results indicate that CTL activity was more critical to recovery from rickettsial infection than were the effects of IFN-γ. The observation that perforin gene knockout mice were more than 100-fold more susceptible than wild-type C57BL/6 mice indicates that perforin-mediated activity accounts for a large component, but not all, of the CTL-mediated antirickettsial effect. CTL activity was expressed by immune CD8 T lymphocytes. Adoptive transfer of immune CD8 T lymphocytes from IFN-γ gene knockout mice intoR. australis-infected IFN-γ gene knockout mice dramatically reduced the infectious rickettsial content in the organs, confirming that CD8 T lymphocytes provide immunity against rickettsiae besides that provided by the secretion of IFN-γ. CTLs appear to be crucial to recovery from rickettsial infection.
Chlamydia pneumoniaeMajor Outer Membrane Protein Is a Surface-Exposed Antigen That Elicits Antibodies Primarily Directed against Conformation-Dependent Determinants
Authors: Wolf Katerina , Fischer Elizabeth , Mead David , Zhong Guangming , Peeling Roseanna , Whitmire Bill , Caldwell Harlan D.
Published: July 2002

The major outer membrane protein (MOMP) ofChlamydia trachomatisserovariants is known to be an immunodominant surface antigen. Moreover, it is known that theC. trachomatisMOMP elicits antibodies that recognize both linear and conformational antigenic determinants. In contrast, it has been reported that the MOMP ofChlamydia pneumoniaeis not surface exposed and is immunorecessive. We hypothesized that the discrepancies betweenC. trachomatisandC. pneumoniaeMOMP exposure on intact chlamydiae and immunogenic properties might be because the focus of the host's immune response is directed to conformational epitopes of theC. pneumoniaeMOMP. We therefore conducted studies aimed at defining the surface exposure of MOMP and the conformational dominance of MOMP antibodies. We present here a description ofC. pneumoniaespecies-specific monoclonal antibody (MAb), GZD1E8, which recognizes a conformational epitope on the surface ofC. pneumoniae. This MAb is potent in the neutralization ofC. pneumoniaeinfectivity in vitro. Another previously describedC. pneumoniaespecies-specific monoclonal antibody, RR-402, displayed very similar characteristics. However, the antigenic determinant recognized by RR-402 has yet to be identified. We show by immunoprecipitation ofC. pneumoniaewith GZD1E8 and RR-402 MAbs and by mass spectrometry analysis of immunoprecipitated proteins that both antibodies GZD1E8 and RR-402 recognize the MOMP ofC. pneumoniaeand that this protein is localized on the surface of the organism. We also show that human sera fromC. pneumoniae-positive donors consistently recognize the MOMP by immunoprecipitation, indicating that the MOMP ofC. pneumoniaeis an immunogenic protein. These findings have potential implications for bothC. pneumoniaevaccine and diagnostic assay development.
Stable Transfection of the BovineNRAMP1Gene into Murine RAW264.7 Cells: Effect onBrucella abortusSurvival
Authors: Barthel Robert , Feng Jianwei , Piedrahita Jorge A. , McMurray David N. , Templeton Joe W. , Adams L. Garry
Published: July 2002

Genetically based natural resistance to brucellosis in cattle provides for novel strategies to control zoonotic diseases. BovineNRAMP1, the homologue of a murine gene (Bcg), has been identified as a major candidate for controlling the in vivo resistant phenotype. We developed an in vitro model for expression of resistance- and susceptibility-associated alleles of bovineNRAMP1as stable transgenes under the regulatory control of the bovineNRAMP1promoter in the murine RAW264.7 macrophage cell line (Bcgs) to analyze the regulation of theNRAMP1gene and its role in macrophage function. We demonstrated that the 5′-flanking region of bovineNRAMP1, despite the lack of TATA and CAAT boxes, has a functional promoter capable of driving the expression of a transgene in murine macrophages. A polymorphism within a microsatellite in the 3′ untranslated region critically affects the expression of bovineNRAMP1and the control of in vitro replication ofBrucella abortusbut notSalmonella entericaserovar Dublin. We did not observe any differences in the production of NO by resting or gamma interferon (IFN-γ)- and IFN-γ–lipopolysaccharide (LPS)-treated transfected cell lines, yet the resistant transfected cell lines produced significantly less NO than other cell lines, following stimulation with LPS at 24 and 48 h.
Immunohistochemical Analysis of Lyme Disease in the Skin of Naive and Infection-Immune Rabbits following Challenge
Authors: Chong-Cerrillo Celeste , Shang Ellen S. , Blanco David R. , Lovett Michael A. , Miller James N.
Published: July 2002

In this study, skin histopathology from naive and infection-derived immune rabbits was compared following intradermal challenge usingBorrelia burgdorferiB31 strain. The presence or absence of spirochetes in relationship to host cellular immune responses was determined from the time of intradermal inoculation to the time of erythema migrans (EM) development (∼7 days in naive rabbits) and through development of challenge immunity (∼5 months in naive rabbits). Skin biopsies were obtained and analyzed for the presence of spirochetes, B cells, T cells, polymorphonuclear cells (PMNs), and macrophages by immunohistochemical techniques. In infected naive animals, morphologically identifiable spirochetes were detected at 2 h and up to 3 weeks postinfection. At 12 and 24 h postinfection there was a marked PMN response that decreased by 36 to 48 h; by 72 h the PMNs were replaced by a few infiltrating macrophages. At the time of EM development and 14 days postinfection, the PMNs and macrophages were replaced by a lymphocytic infiltrate. There was a greater number of spirochetes at 14 days, a time when EM had resolved, than at 7 days postinfection. By 3 weeks postinfection there were few organisms and lymphocytes detectable. In contrast to infected naive rabbits, intact spirochetes were never visualized in skin biopsies from infection-immune rabbits; only spirochetal antigen was detected at 2, 12, and 24 h in the presence of a numerous PMN infiltrate. By 36 h postchallenge, spirochetal antigen could not be detected and the PMN response was replaced by a few infiltrating macrophages. By 72 h postchallenge, PMNs and macrophages were absent from the skin; B and T cells were never detected at any time point in skin from infection-immune rabbits. The destruction of spirochetes in immune animals in the presence of PMNs and in the absence of a lymphocytic infiltrate suggests that infection-derived immunity is antibody mediated.
Role of the Heat Shock Protein 90 in Immune Response Stimulation by Bacterial DNA and Synthetic Oligonucleotides
Authors: Zhu Fu-Gang , Pisetsky David S.
Published: July 2002

To elucidate the mechanisms of immunostimulation by bacterial DNA and synthetic oligonucleotides, the effects of heat shock protein 90 (Hsp90) inhibitors on the activation of murine spleen cells and macrophages by these molecules were investigated. Murine spleen cells and J774 and RAW264.7 macrophages responded to a CpG-containing oligodeoxynucleotide (CpG ODN) andEscherichia coliDNA by increased production of interleukin 6 (IL-6), IL-12, tumor necrosis factor alpha, and nitric oxide (NO). Pretreatment with any of the three Hsp90 inhibitors geldanamycin, radicicol, and herbimycin A resulted in a dose-dependent suppression of cytokine production from the spleen cells and macrophages and of NO from macrophages stimulated with CpG ODN orE. coliDNA. These Hsp90 inhibitors, however, had no effect onStaphylococcus aureusCowan strain 1-induced IL-12 production from either the murine spleen cells or macrophages. CpG ODN andE. coliDNA induced increased intracellular levels of phosphorylated extracellular signal-regulated kinases (ERK1 and -2), which are members of the mitogen-activated protein (MAP) kinase family, while geldanamycin and radicicol blocked the phosphorylation of ERK1 and -2 in J774 and RAW264.7 cells. These data indicate that DNA-induced activation of murine spleen cells and macrophages is mediated by Hsp90 and that Hsp90 inhibitor suppression of DNA-induced macrophage activation is associated with disruption of the MAP kinase signaling pathway. Our findings suggest that Hsp90 inhibitors may provide a useful means of elucidating the mechanisms of immunostimulation by bacterial DNA and CpG ODN as well as a strategy for preventing adverse effects of bacterial DNA as well as lipopolysaccharide.
Dependence of
Mycobacterium bovis
BCG on Anaerobic Nitrate Reductase for Persistence Is Tissue Specific
Authors: Fritz Christian , Maass Silvia , Kreft Andreas , Bange Franz-Christoph
Published: July 2002

Mycobacterium bovis
BCG, the only presently available vaccine against tuberculosis, was obtained from virulent
M. bovis
after serial passages in vitro. The vaccine strain retained at least some of its original virulence, as it persists in immune-competent hosts and occasionally may cause fatal disease in immune-deficient hosts. Mycobacterial persistence in vivo is thought to depend on anaerobic metabolism, an apparent paradox since all mycobacteria are obligate aerobes. Here we report that
M. bovis
BCG lacking anaerobic nitrate reductase (NarGHJI), an enzyme essential for nitrate respiration, failed to persist in the lungs, liver, and kidneys of immune-competent (BALB/c) mice. In immune-deficient (SCID) mice, however, bacilli caused chronic infection despite disruption of
narG
, even if growth of the mutant was severely impaired in lungs, liver, and kidneys. Persistence and growth of BCG in the spleens of either mouse strain appeared largely unaffected by lack of anaerobic nitrate reductase, indicating that the role of the enzyme in pathogenesis is tissue specific. These data suggest first that anaerobic nitrate reduction is essential for metabolism of
M. bovis
BCG in immune-competent but not immune-deficient mice and second that its role in mycobacterial disease is tissue specific, both of which are observations with important implications for pathogenesis of mycobacteria and vaccine development.
The Cytochrome
c
Maturation Locus of
Legionella pneumophila
Promotes Iron Assimilation and Intracellular Infection and Contains a Strain-Specific Insertion Sequence Element
Authors: Viswanathan V. K. , Kurtz Sherry , Pedersen Lisa L. , Abu Kwaik Yousef , Krcmarik Kevin , Mody Sejal , Cianciotto Nicholas P.
Published: July 2002

Previously, we obtained a
Legionella pneumophila
mutant, NU208, that is hypersensitive to iron chelators when grown on standard
Legionella
media. Here, we demonstrate that NU208 is also impaired for growth in media that simply lack their iron supplement. The mutant was not, however, impaired for the production of legiobactin, the only known
L. pneumophila
siderophore. Importantly, NU208 was also highly defective for intracellular growth in human U937 cell macrophages and
Hartmannella
and
Acanthamoeba
amoebae. The growth defect within macrophages was exacerbated by treatment of the host cells with an iron chelator. Sequence analysis demonstrated that the transposon disruption in NU208 lies within an open reading frame that is highly similar to the cytochrome
c
maturation gene,
ccmC
. CcmC is generally recognized for its role in the heme export step of cytochrome biogenesis. Indeed, NU208 lacked cytochrome
c
. Phenotypic analysis of two additional, independently derived
ccmC
mutants confirmed that the growth defect in low-iron medium and impaired infectivity were associated with the transposon insertion and not an entirely spontaneous second-site mutation.
trans
-complementation analysis of NU208 confirmed that
L. pneumophila ccmC
is required for cytochrome
c
production, growth under low-iron growth conditions, and at least some forms of intracellular infection. Although
ccm
genes have recently been implicated in iron assimilation, our data indicate, for the first time, that a
ccm
gene can be required for bacterial growth in an intracellular niche. Complete sequence analysis of the
ccm
locus from strain 130b identified the genes
ccmA-H
. Interestingly, however, we also observed that a 1.8-kb insertion sequence element was positioned between
ccmB
and
ccmC
. Southern hybridizations indicated that the open reading frame within this element (ISLp
1
) was present in multiple copies in some strains of
L. pneumophila
but was absent from others. These findings represent the first evidence for a transposable element in
Legionella
and the first identification of an
L. pneumophila
strain-specific gene.