bluuebunny/crossref_metadata_2025 huggingface dataset:
Pharmacodynamic Activity and Efficacy of Linezolid in a Rat Model of Pneumococcal Pneumonia
Authors: Gentry-Nielsen Martha J. , Olsen Keith M. , Preheim Laurel C.
Published: July 2002

Linezolid is a new oxazolidinone antibiotic with potent activity against gram-positive bacteria, including
Streptococcus pneumoniae
. The pharmacodynamic activity and in vivo efficacy of linezolid were compared to those of ceftriaxone in an immunocompetent rat model of pneumococcal pneumonia. Rats infected intratracheally with 8 × 10
7
CFU of a penicillin-sensitive (MIC, 0.032 μg/ml) strain of
S. pneumoniae
were treated for 5 days beginning 18 h postinfection. Groups of rats were sham treated with oral phosphate-buffered saline or received oral liquid linezolid at 25 or 50 mg/kg of body weight twice a day (b.i.d.) or subcutaneous ceftriaxone at 100 mg/kg once daily. Mortality was monitored for 10 days postinfection; blood culturing was performed on day 1 (pretreatment) and on days 3, 5, and 10 postinfection for the determination of bacteremia. Serum also was collected for the determination of pharmacokinetic and pharmacodynamic parameters at 30 min and at 3, 5, and 12 h (linezolid) or 3, 5, and 24 h (ceftriaxone) postdose. The cumulative mortality rates were 100% for the sham-treated group, 58.3% for the low-dose linezolid group, 8.3% for the high-dose linezolid group, and 0% for the ceftriaxone group. Rats in each of the antibiotic treatment groups had significantly fewer bacteria (
P
< 0.00001) in their bronchoalveolar lavage fluid (BALF) on day 3 postinfection than sham-treated rats. There also were significantly fewer organisms in the BALF of rats treated with ceftriaxone than in the BALF of rats treated with either dose of linezolid. Oral linezolid at 50 mg/kg b.i.d. therefore was as effective as ceftriaxone in experimental pneumococcal pneumonia, whereas the 25-mg/kg b.i.d. dose was significantly less effective. All pharmacodynamic parameters reflected efficacy and were significantly different for the two dosage regimens of linezolid (
P
< 0.01). However, the free-fraction pharmacodynamic parameters predictive of outcome were a value of >39% for the percentage of time in the experimental dosing interval during which the linezolid concentration exceeded the MIC and a value of >147 for the ratio of the area under the serum concentration-time curve to the MIC.
PCR Detection of
Salmonella enterica
Serotype Montevideo in and on Raw Tomatoes Using Primers Derived from
hilA
Authors: Guo Xuan , Chen Jinru , Beuchat Larry R. , Brackett Robert E.
Published: July 2002

Salmonellae have been some of the most frequently reported etiological agents in fresh-produce-associated outbreaks of human infections in recent years. PCR assays using four innovative pairs of primers derived from
hilA
and
sirA
, positive regulators of
Salmonella
invasive genes, were developed to identify
Salmonella enterica
serotype Montevideo on and in tomatoes. Based on examination of 83
Salmonella
strains and 22 non-
Salmonella
strains, we concluded that a pair of
hilA
primers detects
Salmonella
specifically. The detection limits of the PCR assay were 10
1
and 10
0
CFU/ml after enrichment at 37°C for 6 and 9 h, respectively. When the assay was validated by detecting
S. enterica
serotype Montevideo in and on artificially inoculated tomatoes, 10
2
and 10
1
CFU/g were detected, respectively, after enrichment for 6 h at 37°C. Our results suggest that the
hilA
-based PCR assay is sensitive and specific, and can be used for rapid detection of Salmonellae in or on fresh produce.
Uptake and Active Efflux of Polycyclic Aromatic Hydrocarbons by
Pseudomonas fluorescens
LP6a
Authors: Bugg Trevor , Foght Julia M. , Pickard Michael A , Gray Murray R.
Published: July 2002

The mechanism of transport of polycyclic aromatic hydrocarbons (PAHs) by
Pseudomonas fluorescens
LP6a, a PAH-degrading bacterium, was studied by inhibiting membrane transport and measuring the resulting change in cellular uptake. Three cultures were used: wild-type LP6a which carried a plasmid for PAH degradation, a transposon mutant lacking the first enzyme in the pathway for PAH degradation, and a cured strain without the plasmid. Washed cells were mixed with aqueous solutions of radiolabelled PAH; then the cells were removed by centrifugation, and the concentrations of PAH in the supernatant and the cell pellet were measured. The change in the pellet and supernatant concentrations after inhibitors of membrane transport (azide, cyanide, or carbonyl cyanide
m
-chlorophenyl hydrazone) were added indicated the role of active transport. The data were consistent with the presence of two conflicting transport mechanisms: uptake by passive diffusion and an energy-driven efflux system to transport PAHs out of the cell. The efflux mechanism was chromosomally encoded. Under the test conditions used, neither uptake nor efflux of phenanthrene by
P. fluorescens
LP6a was saturated. The efflux mechanism showed selectivity since phenanthrene, anthracene, and fluoranthene were transported out of the cell but naphthalene was not.
Cloning, Nucleotide Sequence, and Expression of the Gene Encoding the Bacteriocin Boticin B from
Clostridium botulinum
Strain 213B
Authors: Dineen Sean S. , Bradshaw Marite , Johnson Eric A.
Published: July 2002

Boticin B is a heat-stable bacteriocin produced by
Clostridium botulinum
strain 213B that has inhibitory activity against various strains of
C. botulinum
and related clostridia. The gene encoding the bacteriocin was localized to a 3.0-kb
Hin
dIII fragment of an 18.8-kb plasmid, cloned, and sequenced. DNA sequencing revealed the boticin B structural gene,
btcB
, to be an open reading frame encoding 50 amino acids. A
C. botulinum
strain 62A transconjugant containing the
Hin
dIII fragment inserted into a clostridial shuttle vector expressed boticin B, although at much lower levels than those observed in
C. botulinum
213B. To our knowledge, this is the first demonstration and characterization of a bacteriocin from toxigenic group I
C. botulinum
.
Ferrous Iron-Dependent Volatilization of Mercury by the Plasma Membrane of
Thiobacillus ferrooxidans
Authors: Iwahori Kenji , Takeuchi Fumiaki , Kamimura Kazuo , Sugio Tsuyoshi
Published: July 2002

Of 100 strains of iron-oxidizing bacteria isolated,
Thiobacillus ferrooxidans
SUG 2-2 was the most resistant to mercury toxicity and could grow in an Fe
2+
medium (pH 2.5) supplemented with 6 μM Hg
2+
. In contrast,
T. ferrooxidans
AP19-3, a mercury-sensitive
T. ferrooxidans
strain, could not grow with 0.7 μM Hg
2+
. When incubated for 3 h in a salt solution (pH 2.5) with 0.7 μM Hg
2+
, resting cells of resistant and sensitive strains volatilized approximately 20 and 1.7%, respectively, of the total mercury added. The amount of mercury volatilized by resistant cells, but not by sensitive cells, increased to 62% when Fe
2+
was added. The optimum pH and temperature for mercury volatilization activity were 2.3 and 30°C, respectively. Sodium cyanide, sodium molybdate, sodium tungstate, and silver nitrate strongly inhibited the Fe
2+
-dependent mercury volatilization activity of
T. ferrooxidans
. When incubated in a salt solution (pH 3.8) with 0.7 μM Hg
2+
and 1 mM Fe
2+
, plasma membranes prepared from resistant cells volatilized 48% of the total mercury added after 5 days of incubation. However, the membrane did not have mercury reductase activity with NADPH as an electron donor. Fe
2+
-dependent mercury volatilization activity was not observed with plasma membranes pretreated with 2 mM sodium cyanide. Rusticyanin from resistant cells activated iron oxidation activity of the plasma membrane and activated the Fe
2+
-dependent mercury volatilization activity of the plasma membrane.
Murine Monoclonal Antibodies Specific for Lipopolysaccharide ofEscherichia coliO26 and O111
Authors: Rivera-Betancourt Mildred , Keen James E.
Published: July 2002

Monoclonal antibody (MAb) 12F5 reacted with 35Escherichia coliO26 isolates and cross-reacted with 1 of 365 non-E. coliO26 isolates. MAb 15C4 reacted with 30E. coliO111 strains and 8SalmonellaO35 strains (possessing identical O antigen) but not with 362 other bacterial strains. Lipopolysaccharide immunoblots confirmed MAb O-antigen specificity.
Degradation of Quercetin and Luteolin byEubacterium ramulus
Authors: Braune Annett , Gütschow Michael , Engst Wolfram , Blaut Michael
Published: July 2002

The degradation of the flavonol quercetin and the flavone luteolin byEubacterium ramulus, a strict anaerobe of the human intestinal tract, was studied. Resting cells converted these flavonoids to 3,4-dihydroxyphenylacetic acid and 3-(3,4-dihydroxyphenyl)propionic acid, respectively. The conversion of quercetin was accompanied by the transient formation of two intermediates, one of which was identified as taxifolin based on its specific retention time and UV and mass spectra. The structure of the second intermediate, alphitonin, was additionally elucidated by1H and13C nuclear magnetic resonance analysis. In resting-cell experiments, taxifolin in turn was converted via alphitonin to 3,4-dihydroxyphenylacetic acid. Alphitonin, which was prepared by enzymatic conversion of taxifolin and subsequent purification, was also transformed to 3,4-dihydroxyphenylacetic acid. The coenzyme-independent isomerization of taxifolin to alphitonin was catalyzed by cell extract or a partially purified enzyme preparation ofE. ramulus. The degradation of luteolin by resting cells ofE. ramulusresulted in the formation of the intermediate eriodictyol, which was identified by high-performance liquid chromatography and mass spectrometry analysis. The observed intermediates of quercetin and luteolin conversion suggest that the degradation pathways inE. ramulusstart with an analogous reduction step followed by different enzymatic reactions depending on the additional 3-hydroxyl group present in the flavonol structure.
Characterization and Pathogenic Potential ofListeria monocytogenesIsolates from the Smoked Fish Industry
Authors: Norton Dawn M. , Scarlett Janet M. , Horton Kelly , Sue David , Thimothe Joanne , Boor Kathryn J. , Wiedmann Martin
Published: July 2002

This study was designed to evaluate the hypothesis that some of theListeria monocytogenessubtypes associated with foods, specifically smoked fish, may have an attenuated ability to cause human disease. We tested this hypothesis by using two different approaches: (i) comparison of molecular subtypes found among 117 isolates from smoked fish, raw materials, fish in process, and processing environments with subtypes found among a collection of 275 human clinical isolates and (ii) the evaluation of the cytopathogenicity of industrial isolates. Ribotyping and PCR-restriction fragment length polymorphism typing of thehlyAandactAgenes differentiated 23 subtypes among the industrial isolates and allowed classification of the isolates into three genetic lineages. A significantly higher proportion of human isolates (69.1%) than industrial isolates (36.8%) were classified as lineage I, which contains human sporadic isolates and all epidemic isolates. All other industrial isolates (63.2%) were classified as lineage II, which contains only human sporadic isolates. Lineage I ribotypes DUP-1038B and DUP-1042B represented a significantly higher proportion of the human isolates than industrial isolates (5.1%). Lineage II ribotypes DUP-1039C, DUP-1042C, and DUP-1045, shown previously to persist in the smoked fish processing environment, represented nearly 50% of the industrial isolates, compared to 7.6% of the human isolates. Representatives of each subtype were evaluated with a tissue culture plaque assay. Lineage I isolates formed plaques that were significantly larger than those formed by lineage II isolates. Isolates from the smoked fish industry representing three ribotypes formed no plaques or small plaques, indicating that they had an impaired ability to infect mammalian cells. WhileL. monocytogenesclonal groups linked to human listeriosis cases and outbreaks were isolated, our data also suggest that at least someL. monocytogenessubtypes present in ready-to-eat foods may have limited human-pathogenic potential.
Characterization of Recurrent and Sporadic
Listeria monocytogenes
Isolates from Raw Milk and Nondairy Foods by Pulsed-Field Gel Electrophoresis, Monocin Typing, Plasmid Profiling, and Cadmium and Antibiotic Resistance Determination
Authors: Harvey J. , Gilmour A.
Published: July 2002

Following previous surveys to assess the incidence of
Listeria monocytogenes
in raw milk and nondairy foods processed in Northern Ireland, isolates were characterized as recurrent or sporadic on the basis of multilocus enzyme electrophoresis (MEE) analysis and restriction fragment length polymorphism typing. In the present study, 45 representative recurrent and sporadic electrophoretic types (ETs) previously identified by MEE were subjected to pulsed-field gel electrophoresis (PFGE) of genomic DNA macrorestriction fragments, monocin typing, plasmid profiling, and an examination of resistance to cadmium and nine different antibiotics. Although PFGE proved to be capable of subdividing a number of recurrent and sporadic ETs, the grouping of strains arrived at by PFGE and MEE were in broad agreement, and previous conclusions regarding the designation of
L. monocytogenes
strains as recurrent or sporadic remained unaltered. It is considered that PFGE was able to detect minor genetic changes in recurrent ETs which occurred during the time period in which food surveys were carried out. Production of type E monocin (Types A to E were found among the 45 strains), plasmid carriage, and resistance to cadmium occurred more frequently in recurrent than in sporadic strains and may be important with regard to the ability of
L. monocytogenes
to persist in food and food-processing environments. Only 2 of 45 strains showed resistance to any of the nine antibiotics tested: two sporadic strains were resistant to tetracycline (MIC, 64 μg ml
−1
).
Metabolism of Benzoate, Cyclohex-1-ene Carboxylate, and Cyclohexane Carboxylate by “
Syntrophus aciditrophicus
” Strain SB in Syntrophic Association with H
2
-Using Microorganisms
Authors: Elshahed Mostafa S. , Bhupathiraju Vishvesh K. , Wofford Neil Q. , Nanny Mark A. , McInerney Michael J.
Published: July 2002

The metabolism of benzoate, cyclohex-1-ene carboxylate, and cyclohexane carboxylate by “
Syntrophus aciditrophicus
” in cocultures with hydrogen-using microorganisms was studied. Cyclohexane carboxylate, cyclohex-1-ene carboxylate, pimelate, and glutarate (or their coenzyme A [CoA] derivatives) transiently accumulated during growth with benzoate. Identification was based on comparison of retention times and mass spectra of trimethylsilyl derivatives to the retention times and mass spectra of authentic chemical standards.
13
C nuclear magnetic resonance spectroscopy confirmed that cyclohexane carboxylate and cyclohex-1-ene carboxylate were produced from [
ring
-
13
C
6
]benzoate. None of the metabolites mentioned above was detected in non-substrate-amended or heat-killed controls. Cyclohexane carboxylic acid accumulated to a concentration of 260 μM, accounting for about 18% of the initial benzoate added. This compound was not detected in culture extracts of
Rhodopseudomonas palustris
grown phototrophically or
Thauera aromatica
grown under nitrate-reducing conditions. Cocultures of “
S. aciditrophicus
” and
Methanospirillum hungatei
readily metabolized cyclohexane carboxylate and cyclohex-1-ene carboxylate at a rate slightly faster than the rate of benzoate metabolism. In addition to cyclohexane carboxylate, pimelate, and glutarate, 2-hydroxycyclohexane carboxylate was detected in trace amounts in cocultures grown with cyclohex-1-ene carboxylate. Cyclohex-1-ene carboxylate, pimelate, and glutarate were detected in cocultures grown with cyclohexane carboxylate at levels similar to those found in benzoate-grown cocultures. Cell extracts of “
S. aciditrophicus
” grown in a coculture with
Desulfovibrio
sp. strain G11 with benzoate or in a pure culture with crotonate contained the following enzyme activities: an ATP-dependent benzoyl-CoA ligase, cyclohex-1-ene carboxyl-CoA hydratase, and 2-hydroxycyclohexane carboxyl-CoA dehydrogenase, as well as pimelyl-CoA dehydrogenase, glutaryl-CoA dehydrogenase, and the enzymes required for conversion of crotonyl-CoA to acetate. 2-Ketocyclohexane carboxyl-CoA hydrolase activity was detected in cell extracts of “
S. aciditrophicus
”-
Desulfovibrio
sp. strain G11 benzoate-grown cocultures but not in crotonate-grown pure cultures of “
S. aciditrophicus
”. These results are consistent with the hypothesis that ring reduction during syntrophic benzoate metabolism involves a four- or six-electron reduction step and that once cyclohex-1-ene carboxyl-CoA is made, it is metabolized in a manner similar to that in
R. palustris
.
Lack of Cross-Resistance to Cry19A from
Bacillus thuringiensis
subsp.
jegathesan
in
Culex quinquefasciatus
(Diptera: Culicidae) Resistant to Cry Toxins from
Bacillus thuringiensis
subsp.
israelensis
Authors: Wirth Margaret C. , Delécluse Armelle , Walton William E.
Published: July 2002

Culex quinquefasciatus
mosquitoes with high levels of resistance to single or multiple toxins from
Bacillus thuringiensis
subsp.
israelensis
were tested for cross-resistance to the
Bacillus thuringiensis
subsp.
jegathesan
polypeptide Cry19A. No cross-resistance was detected in mosquitoes that had been selected with the Cry11A, Cry4A and Cry4B, or Cry4A, Cry4B, Cry11A, and CytA toxins. A low but statistically significant level of cross-resistance, three to fourfold, was detected in the colony selected with Cry4A, Cry4B, and Cry11A. This cross-resistance was similar to that previously detected with
B. thuringiensis
subsp.
jegathesan
in the same colony. These data help explain the toxicity of
B. thuringiensis
subsp.
jegathesan
against the resistant colonies and indicate that the Cry19A polypeptide might be useful in managing resistance and/or as a component of synthetic combinations of mosquitocidal toxins.
Detection of Legionellae in Hospital Water Samples by Quantitative Real-Time LightCycler PCR
Authors: Wellinghausen Nele , Frost Cathrin , Marre Reinhard
Published: July 2002

Contamination of hospital water systems with legionellae is a well-known cause of nosocomial legionellosis. We describe a new real-time LightCycler PCR assay for quantitative determination of legionellae in potable water samples. Primers that amplify both a 386-bp fragment of the 16S rRNA gene from
Legionella
spp. and a specifically cloned fragment of the phage lambda, added to each sample as an internal inhibitor control, were used. The amplified products were detected by use of a dual-color hybridization probe assay design and quantified with external standards composed of
Legionella pneumophila
genomic DNA. The PCR assay had a sensitivity of 1 fg of
Legionella
DNA (i.e., less than one
Legionella
organism) per assay and detected 44
Legionella
species and serogroups. Seventy-seven water samples from three hospitals were investigated by PCR and culture. The rates of detection of legionellae were 98.7% (76 of 77) by the PCR assay and 70.1% (54 of 77) by culture; PCR inhibitors were detected in one sample. The amounts of legionellae calculated from the PCR results were associated with the CFU detected by culture (
r
= 0.57;
P
< 0.001), but PCR results were mostly higher than the culture results. Since
L. pneumophila
is the main cause of legionellosis, we further developed a quantitative
L. pneumophila
-specific PCR assay targeting the macrophage infectivity potentiator (
mip)
gene, which codes for an immunophilin of the FK506 binding protein family. All but one of the 16S rRNA gene PCR-positive water samples were also positive in the
mip
gene PCR, and the results of the two PCR assays were correlated. In conclusion, the newly developed
Legionella
genus-specific and
L. pneumophila
species-specific PCR assays proved to be valuable tools for investigation of
Legionella
contamination in potable water systems.
Use of Multiplex PCR and PCR Restriction Enzyme Analysis for Detection and Exploration of the Variability in the Free-Living AmoebaNaegleriain the Environment
Authors: Pélandakis Michel , Pernin Pierre
Published: July 2002

A multiplex PCR was developed to simultaneously detectNaegleria fowleriand otherNaegleriaspecies in the environment. Multiplex PCR was also capable of identifyingN. fowleriisolates with internal transcribed spacers of different sizes. In addition, restriction fragment length polymorphism analysis of the PCR product distinguished the main thermophilicNaegleriaspecies from the sampling sites.
Phagocytic Function of Monocytes in Children with Human Immunodeficiency Virus Type 1 Infection
Authors: Tardei Gratiela , Duiculescu Dan , Capo Christian , Diaconu Carmen-Cristina , Mutiu Adrian , Mege Jean-Louis , Cernescu Costin Eugen
Published: July 2002

We investigated the phagocytic function of monocytes in 7- to 10-year-old children horizontally infected with human immunodeficiency virus type 1 (HIV-1) in comparison to that in healthy sex- and age-matched controls. CR3-mediated phagocytosis was increased in patients with HIV-associated pulmonary tuberculosis, independently of CD4 counts and p24 antigenemia.
Immunological Characterization of
Escherichia coli
O157:H7 Intimin γ1
Authors: Son W.-G. , Graham T. A. , Gannon V. P. J.
Published: July 2002

Portions of the intimin genes of
Escherichia coli
O157:H7 strain E319 and of the enteropathogenic
E. coli
O127:H6 strain E2348/69 were amplified by PCR and cloned into pET-28a(+) expression vectors. The entire 934 amino acids (aa) of
E. coli
O157:H7 intimin, the C-terminal 306 aa of
E. coli
O157:H7 intimin, and the C-terminal 311 aa of
E. coli
O127:H6 intimin were expressed as proteins fused with a six-histidine residue tag (six-His tag) in pET-28a(+). Rabbit antisera raised against the six-His tag-full-length
E. coli
O157:H7 intimin protein fusion cross-reacted in slot and Western blots with outer membrane protein preparations from the majority of enterohemorrhagic and enteropathogenic
E. coli
serotypes which have the intimin gene. The
E. coli
strains tested included isolates from humans and animals which produce intimin typesα (O serogroups 86, 127, and 142), β1 (O serogroups 5, 26, 46, 69, 111, 126, and 128), γ1 (O serogroups 55, 145, and 157), γ2 (O serogroups 111 and 103), and ε (O serogroup 103) and a nontypeable intimin (O serogroup 80), results based on intimin type-specific PCR assays. Rabbit antisera raised against the
E. coli
O157:H7 C-terminal fusion protein were much more intimin type-specific than those raised against the full-length intimin fusion protein, but some cross-reaction with other intimin types was also observed for these antisera. In contrast, the monoclonal antibody Intγ1.C11, raised against the C-terminal
E. coli
O157 intimin, reacted only with preparations from intimin γ1-producing
E. coli
strains such as
E. coli
O157:H7.
Evaluation of Enzyme-Linked Immunosorbent Assay for Diagnosis of Post-Kala-Azar Dermal Leishmaniasis with Crude or Recombinant k39 Antigen
Authors: Salotra P. , Sreenivas G. , Nasim A. A. , Subba Raju B. V. , Ramesh V.
Published: July 2002

The diagnosis of post-kala-azar dermal leishmaniasis (PKDL), a dermatosis that provides the only known reservoir for the parasite
Leishmania donovani
in India, remains a problem. Timely recognition and treatment of PKDL would contribute significantly to the control of kala-azar. We evaluated here the potential of the enzyme-linked immunosorbent assay (ELISA) as a diagnostic tool for PKDL. Antigen prepared from promastigotes and axenic amastigotes with parasite isolates that were derived from skin lesions of a PKDL patient gave sensitivities of 86.36 and 92%, respectively, in the 88 PKDL cases examined. The specificity of the ELISA test was examined by testing groups of patients with other skin disorders (leprosy and vitiligo) or coendemic infections (malaria and tuberculosis), as well as healthy controls from areas where this disease is endemic or is not endemic. A false-positive reaction was obtained in 14 of 144 (9.8%) of the controls with the promastigote antigen and in 14 of 145 (9.7%) of the controls with the amastigote antigen. Evaluation of the serodiagnostic potential of recombinant k39 by ELISA revealed a higher sensitivity (94.5%) and specificity (93.7%) compared to the other two antigens used. The data demonstrate that ELISA with crude or recombinant antigen k39 provides a relatively simple and less-invasive test for the reliable diagnosis of PKDL.
Effect of Exogenous Interleukin-18 (IL-18) and IL-12 in the Course ofBrucella abortus2308 Infection in Mice
Authors: Pasquali Paolo , Adone Rosanna , Gasbarre Louis C. , Pistoia Claudia , Ciuchini Franco
Published: July 2002

In this study we demonstrated that combined inoculation of interleukin-12 (IL-12) and IL-18 reduced the number of bacteria in the spleens of mice infected withBrucella abortus2308 and that the effect of the treatment was mediated by an increased capability of spleen cells to produce gamma interferon at the early phase of infection.
Activation of Human NK Cells by Staphylococci and Lactobacilli Requires Cell Contact-Dependent Costimulation by Autologous Monocytes
Authors: Haller D. , Serrant P. , Granato D. , Schiffrin E. J. , Blum S.
Published: July 2002

NK cells are instrumental in innate immune responses, in particular for the early production of gamma interferon (IFN-γ) and other cytokines necessary to control certain bacterial, parasitic, and viral infections. NK cell-mediated effector functions are controlled by a fine balance between distinct receptors mediating activating and inhibitory signals; however, little is known about activating receptors on NK cells and their corresponding ligands. Several studies have shown that commensal lactobacilli isolated from the human gastrointestinal tract activate human mononuclear cells and are potent inducers of IFN-γ and monocyte-derived interleukin 12 (IL-12). NK cell activation was shown forLactobacillus johnsoniiLa1. In this study the cellular mechanisms of in vitro NK cell activation by gram-positive bacteria were analyzed.Staphylococcus aureus- andL. johnsoniiLa1-mediated activation of CD3−CD16+CD56+human peripheral blood NK cells, including expression of the activation antigen CD69 and secretion of IFN-γ, required cell contact-dependent costimulation by autologous monocytes.S. aureus-andL. johnsonii-preactivated monocytes retained their capacity to induce NK cell activation. In contrast, cytokine-primed monocytes completely failed to induce NK cell activation unless bacteria were present. This suggests that phagocytosis of bacteria provided additional coactivation signals on accessory cells that may differ from those induced by tumor necrosis factor and IFN-γ. Blocking of costimulatory molecules by B7.1, B7.2, and IL-12 but not CD14 monoclonal antibodies inhibitedS. aureus- andL. johnsonii-induced effector function of NK cells. Our data suggest an important role for accessory cell-derived signals in the process of NK cell activation by gram-positive bacteria.
Recombinant Bovine Interleukin-1β Amplifies the Effects of Partially PurifiedPasteurella haemolyticaLeukotoxin on Bovine Neutrophils in a β2-Integrin-Dependent Manner
Authors: Leite F. , Brown J. F. , Sylte M. J. , Briggs R. E. , Czuprynski C. J.
Published: July 2002

The influx and death of polymorphonuclear leukocytes within the infected lung are hallmarks of bovine pasteurellosis. Recent reports have shown that thePasteurella haemolyticaleukotoxin (LKT) and other RTX toxins bind β2-integrins on target cells. In this study we demonstrate that exposure of bovine neutrophils to recombinant bovine interleukin-1β upregulates β2-integrins (CD11a/CD18), which in turn enhance the binding and amplify the biological effects of partially purified LKT on these cells. LKT binding and cytotoxicity were inhibited by addition of an anti-integrin antibody (CD11a/CD18). These findings help to clarify the early events that occur in bovine pasteurellosis and support the hypothesis that inflammatory mediators might increase the severity of pasteurellosis by causing upregulation of β2-integrins that serve as an LKT receptor on bovine neutrophils.
Genetic Characterization of DNA Region Containing the
trh
and
ure
Genes of
Vibrio parahaemolyticus
Authors: Park Kwon-Sam , Iida Tetsuya , Yamaichi Yoshiharu , Oyagi Tomohito , Yamamoto Koichiro , Honda Takeshi
Published: July 2002

We have demonstrated that possession of the gene for thermostable direct hemolysin-related hemolysin (
trh
) coincides with the presence of the urease gene among clinical
Vibrio parahaemolyticus
strains and that the location of the two genes are in close proximity on the chromosome. Here, we cloned and sequenced the 15,754-bp DNA region containing the
trh
gene and the gene cluster for urease production from the chromosome of clinical
V. parahaemolyticus
(TH3996). We found 16 open reading frames (ORFs) and a lower G+C content (41%) compared with the total genome of this bacterium (46 to 47%). The
ure
cluster consisted of eight genes, namely,
ureDABCEFG
and
ureR. ureR
was located 5.2 kb upstream of the other seven genes in the opposite direction. The genetic organization and sequences of the
ure
genes resembled those found in
Proteus mirabilis
. Between
ureR
and the other
ure
genes, there were five ORFs, which are homologous with the nickel transport operon (
nik
) of
Escherichia coli
. We disrupted each of the
ureR
,
ureC
, and
nikD
genes in TH3996 by homologous recombination and analyzed the phenotype of the mutants. In the presence of urea these mutant strains had dramatically less urease activity than the strain they were derived from. Disruption of
ureR
,
nikD
, or
ureC
, however, had no effect on TRH production. The DNA region containing the
trh
,
nik
, and
ure
genes was found in only
trh
-positive strains and not in Kanagawa phenomenon-positive and environmental
V. parahaemolyticus
strains. At the end of the region, an insertion sequence-like element existed. These results suggest that the DNA region was introduced into
V. parahaemolyticus
in the past through a mechanism mediated by insertion sequences. This is the first reported case that the genes for an ATP-binding cassette-type nickel transport system, which may play a role in nickel transport through bacterial cytoplasmic membrane, are located adjacent to the
ure
cluster on the genome of an organism.
Identification of Lipoprotein Homologues of Pneumococcal PsaA in the Equine Pathogens
Streptococcus equi
and
Streptococcus zooepidemicus
Authors: Harrington Dean J. , Greated Joanne S. , Chanter Neil , Sutcliffe Iain C.
Published: July 2002

Streptococcus equi
and
Streptococcus zooepidemicus
are major etiological agents of upper and lower airway disease in horses. Despite the considerable animal suffering and economic burden associated with these diseases, the factors that contribute to the virulence of these equine pathogens have not been extensively investigated. Here we demonstrate the presence of a homologue of the
Streptococcus pneumoniae
PsaA protein in both of these equine pathogens. Inhibition of signal peptide processing by the antibiotic globomycin confirmed the lipoprotein nature of the mature proteins, and surface exposure was confirmed by their release from intact cells by mild trypsinolysis.
Dose-Dependent Activation of Lymphocytes in Endotoxin-Induced Airway Inflammation
Authors: Larsson Roland , Rocksén David , Lilliehöök Bo , Jonsson Åsa , Bucht Anders
Published: July 2002

Recruitment of neutrophils to lung tissue and airspaces is a hallmark of inflammatory events following inhalation of endotoxins. We studied the role of different lymphocyte subsets in this inflammation, which is assumed to primarily involve the innate immune system. Inhalation of aerosolizedEscherichia colilipopolysaccharide (LPS) in mice induced a dose-dependent increase in neutrophils in bronchoalveolar lavage fluid, reaching a maximum after 12 h at a low dose and after 24 h at a high dose. Profiles of gene expression in lung tissue indicated an early (2 h) and transient onset of proinflammatory cytokines and chemokines by a low dose of LPS, while a high dose caused more delayed and sustained (6 to 12 h) activation. Gamma interferon, interleukin-2 (IL-2), RANTES, and the α chain of the IL-2 receptor were not expressed at a low dose, whereas a high dose of LPS induced a strong expression of these genes, indicating a dose-dependent activation of T cells. A similar pattern was observed for IL-17, supporting a contribution of T cells to the neutrophilic inflammation only at high-dose exposure to LPS. The involvement of lymphocytes in the inflammatory response was further studied using mice with functional deficiencies in defined lymphocyte subsets. Both γδ T-cell- and B-cell-deficient mice displayed a response similar to that of the corresponding wild-type strains. Selective depletion of NK cells by in vivo administration of the pk136 antibody did not significantly affect the recruitment of neutrophils into airspaces. Thus, neither NK cells, B cells, nor γδ T cells appeared to participate in the host response, suggesting that among the lymphocyte subsets, αβ T cells are exclusively involved in endotoxin-induced airway inflammation.
Cleavage of a Recombinant Human Immunoglobulin A2 (IgA2)-IgA1 Hybrid Antibody by Certain Bacterial IgA1 Proteases
Authors: Senior Bernard W. , Dunlop James I. , Batten Margaret R. , Kilian Mogens , Woof Jenny M.
Published: July 2002

To understand more about the factors influencing the cleavage of immunoglobulin A1 (IgA1) by microbial IgA1 proteases, a recombinant human IgA2/IgA1 hybrid molecule was generated. In the hybrid, termed IgA2/A1 half hinge, a seven-amino-acid sequence corresponding to one half of the duplicated sequence making up the IgA1 hinge was incorporated into the equivalent site in IgA2. Insertion of the IgA1 half hinge into IgA2 did not affect antigen binding capacity or the functional activity of the hybrid molecule, as judged by its ability to bind to IgA Fcα receptors and trigger respiratory bursts in neutrophils. Although the IgA2/A1 hybrid contained only half of the IgA1 hinge, it was found to be cleaved by a variety of different bacterial IgA1 proteases, including representatives of those that cleave IgA1 in the different duplicated halves of the hinge, namely, those ofPrevotella melaninogenica,Streptococcus pneumoniae,S. sanguis,Neisseria meningitidistypes 1 and 2,N. gonorrhoeaetypes 1 and 2, andHaemophilus influenzaetype 2. Thus, for these enzymes the recognition site for IgA1 cleavage is contained within half of the IgA1 hinge region; additional distal elements, if required, are provided by either an IgA1 or an IgA2 framework. In contrast, the IgA2/A1 hybrid appeared to be resistant to cleavage withS. oralisand someH. influenzaetype 1 IgA1 proteases, suggesting these enzymes require additional determinants for efficient substrate recognition.
Priming of a β-Galactosidase (β-GAL)-Specific Type 1 Response in BALB/c Mice Infected with β-GAL-TransfectedLeishmania major
Authors: Chakkalath Hrishekesh R. , Siddiqui Afzal A. , Shankar Anuraj H. , Dobson Deborah E. , Beverley Stephen M. , Titus Richard G.
Published: July 2002

To determine whether an ongoing response toLeishmania majorwould affect the response to a non-cross-reacting, non-leishmanial antigen, susceptible BALB/c mice and resistant C3H mice were infected withL. majorparasites expressingEscherichia coliβ-galactosidase (β-GAL); this parasite was designatedL. major-βGAL. BALB/c and C3H mice responded to infection withL. major-βGALby mounting a CD4 T-cell response to both parasite antigens and to the reporter antigen, β-GAL. The phenotypes of these T cells were characterized after generating T-cell lines from infected mice. As expected, BALB/c mice responded to infection withL. major-βGALby producing interleukin 4 in response to the parasite and C3H mice produced gamma interferon (IFN-γ) in response to the parasite and β-GAL. Interestingly, however, BALB/c mice produced IFN-γ in response to β-GAL. Taken together, these results demonstrate that priming of IFN-γ-producing cells can occur in BALB/c mice despite the fact the animals are simultaneously mounting a potent Th2 response toL. major.
Stability of
Borrelia burgdorferi bdr
Loci In Vitro and In Vivo
Authors: Zückert Wolfram R. , Barbour Alan G.
Published: July 2002

The Lyme disease spirochete
Borrelia burgdorferi
expresses diverse subsurface yet antigenically cross-reactive Bdr protein paralogs from distinct circular- and linear-plasmid loci. We assessed the possible effects of in vitro and in vivo growth on
bdr
locus structure, searching for recombinational events leading to either deletions or insertions of central repeat units or novel amino- and carboxy-terminus combinations. Our data indicate that, apart from plasmid loss during in vitro cultivation, the
bdr
paralog loci of strain B31 are stable. This suggests that recombinatorial variation of
bdr
genes is not essential for persistent mammalian infection.
Chronic Atrophic Gastritis in SCID Mice Experimentally Infected withCampylobacter fetus
Authors: Young Vincent B. , Dangler Charles A. , Fox James G. , Schauer David B.
Published: July 2002

Campylobacter fetusis a cause of enteritis and invasive extraintestinal disease in humans. In order to develop an animal model ofC. fetusinfection, outbred ICR SCID mice were orally challenged with a clinical isolate ofC. fetus. The stomachs of SCID mice were heavily colonized withC. fetus, and colonization was associated with the development of chronic atrophic gastritis. This lesion was characterized by an inflammatory infiltrate of granulocytes and macrophages that over time resulted in a loss of specialized parietal and chief cells in the corpus and the appearance of a metaplastic mucous epithelium. This lesion bears similarity to that encountered during experimental murine infection withHelicobacter pyloriorHelicobacter felis. Despite colonization of the cecum and colon tissues byC. fetusin SCID mice, no lesions were noted in these tissues. A follow-up study confirmed these findings for SCID mice and also demonstrated thatC. fetuscould also infect the gastric mucosa of wild-type, outbred ICR mice. However, in ICR mice, the anatomic extent of colonization was more limited and the severity of inflammation and epithelial alterations was significantly less than that observed in infected SCID mice. The stomach may represent an unrecognized environmental niche forCampylobacterspecies.
Localization of
Haemophilus ducreyi
at the Pustular Stage of Disease in the Human Model of Infection
Authors: Bauer Margaret E. , Spinola Stanley M.
Published: July 2002

To localize
Haemophilus ducreyi
in vivo, human subjects were experimentally infected with
H. ducreyi
until they developed a painful pustule or for 14 days. Lesions were biopsied, and biopsy samples were fixed in 4% paraformaldehyde, and cryosectioned. Sections were stained with polyclonal anti-
H. ducreyi
antiserum or
H. ducreyi
-specific monoclonal antibodies (MAbs) and fluorescently tagged secondary antibodies and examined by confocal microscopy. We identified
H. ducreyi
in 16 of 18 pustules but did not detect bacteria in the one papule examined.
H. ducreyi
was observed as individual cells and in clumps or chains. Staining with MAbs 2D8, 5C9, 3B9, 2C7, and 9D12 demonstrated that
H. ducreyi
expresses the major pilus subunit, FtpA, the 28-kDa outer membrane protein Hlp, the 18-kDa outer membrane protein PAL, and the major outer membrane protein (MOMP) or OmpA2 in vivo. By dual staining with polyclonal anti-
H. ducreyi
antiserum and MAbs that recognize human skin components, we observed bacteria within the neutrophilic infiltrates of all positively staining pustules and in the dermis of 10 of 16 pustules. We were unable to detect bacteria associated with keratinocytes in the samples examined. The data suggest that
H. ducreyi
is found primarily in association with neutrophils and in the dermis at the pustular stage of disease in the human model of infection.
Effect of Preexisting Immunity to
Salmonella
on the Immune Response to Recombinant
Salmonella enterica
Serovar Typhimurium Expressing a
Porphyromonas gingivalis
Hemagglutinin
Authors: Kohler James J. , Pathangey Latha B. , Gillespie Sheila R. , Brown Thomas A.
Published: July 2002

Recombinant
Salmonella
strains expressing foreign heterologous genes have been extensively studied as live oral vaccine delivery vectors. We have investigated the mucosal and systemic immune responses following oral immunization with a recombinant
Salmonella enterica
serovar Typhimurium expressing the hemagglutinin HagB from
Porphyromonas gingivalis
, a suspected etiological agent of adult periodontal disease. We have previously shown a primary mucosal and systemic response following oral immunization with χ4072/pDMD1 and recall responses following boosting at 14 weeks after primary immunization. In this study, we examined the effects of earlier boosting as well as the effects of deliberately induced immunity to the
Salmonella
carrier strain on subsequent immune responses. Mice boosted at week 7 following immunization, a point which corresponded to the peak of the primary response, generally showed lower responses than those boosted at week 14. When mice were preimmunized with the
Salmonella
carrier alone and then immunized with the recombinant strain 7 or 14 weeks later, significant reductions were seen for serum immunoglobulin G (IgG) antibodies at week 14 and for salivary IgA at week 7. No reductions were seen in serum IgA or vaginal wash IgA antibodies. Mice appear to be refractory to boosting with orally administered salmonellae at 7 weeks. Deliberate immunization with the carrier strain did not appreciably affect recall responses at 14 weeks, with the exception of the serum IgG responses, nor did it affect colonization of the Peyer's patches.
Possible Release of an ArgGlyArgProGln Pentapeptide with Innate Immunity Properties from Acidic Proline-Rich Proteins by Proteolytic Activity in Commensal
Streptococcus
and
Actinomyces
Species
Authors: Li Tong , Bratt Per , Jonsson Andreas P. , Ryberg Mats , Johansson Ingegerd , Griffiths William J. , Bergman Tomas , Strömberg Nicklas
Published: July 2002

This study suggests degradation of salivary acidic proline-rich proteins (PRPs) into potential innate-immunity-like peptides by oral
Streptococcus
and
Actinomyces
species. PRP degradation paralleled cleavage of Pro-containing substrates. PRP degradation by
S. gordonii
strain SK12 instantly released a Pyr
1
-Pro
104
Pro
105
and a Gly
111
-Pro
149
Gln
150
peptide together with a presumed Arg
106
Gly
107
Arg
108
Pro
109
Gln
110
pentapeptide. The synthetic Arg
106
Gly
107
Arg
108
Pro
109
Gln
110
peptide desorbed bound bacteria and counteracted sucrose-induced decrease of dental plaque pH in vitro.
Human Resistance toPlasmodium falciparumIncreases during Puberty and Is Predicted by Dehydroepiandrosterone Sulfate Levels
Authors: Kurtis Jonathan D. , Mtalib Ramadhan , Onyango Frederick K. , Duffy Patrick E.
Published: July 2002

Immunity toPlasmodium falciparumdevelops slowly in areas of endemicity, and this is often ascribed to poorly immunogenic or highly variant parasite antigens. However, among populations newly exposed to malaria, adults acquire immunity more rapidly than children. We examined the relationship between pubertal development and resistance toP. falciparum. During two transmission seasons in western Kenya, we treated the same cohort of young males to eradicateP. falciparumand then obtained blood smears each week for 4 months. We determined pubertal development by Tanner staging and by levels of dehydroepiandrosterone sulfate (DHEAS) and testosterone in plasma. In multivariate and age-stratified analyses, we examined the effect of pubertal development on resistance to malaria. In both seasons (n= 248 and 144 volunteers, respectively), older males were less susceptible than younger males. Age-related decreases in the frequency and density of parasitemia were greatest during puberty (15- to 20-year-olds). DHEAS and testosterone were significant independent predictors of resistance toP. falciparumparasitemia, even after accounting for the effect of age. Fifteen- to 20-year-old males with high DHEAS levels had a 72% lower mean parasite density (P< 0.01) than individuals with low DHEAS levels. Similarly, 21- to 35-year-old males with high DHEAS levels had a 92% lower mean parasite density (P< 0.001) and 48% lower frequency of parasitemia (P< 0.05) than individuals with low DHEAS levels. These data suggest that the long period needed to attain full immunity could be explained as a consequence of host development rather than as the requirement to recognize variant or poorly immunogenic parasite antigens.
Cloning and Expression of theActinobacillus actinomycetemcomitansThioredoxin (trx) Gene and Assessment of Cytokine Inhibitory Activity
Authors: Henderson Brian , Tabona Peter , Poole Stephen , Nair Sean P.
Published: July 2002

Thioredoxin is a ubiquitous redox control and cell stress protein. Unexpectedly, in recent years, thioredoxins have been found to exhibit both cytokine and chemokine activities, and there is increasing evidence that this class of protein plays a role in the pathogenesis of inflammatory diseases. In spite of this evidence, it has been reported that the oral bacterium and periodontopathogenActinobacillus actinomycetemcomitanssecretes an immunosuppressive factor (termed suppressive factor 1 [SF1] [T. Kurita-Ochiai and K. Ochiai, Infect. Immun. 64:50–54, 1996]) whose N-terminal sequence, we have determined, identifies it as thioredoxin. We have cloned and expressed the gene encoding the thioredoxin ofA. actinomycetemcomitansand have purified the protein to homogeneity. TheA. actinomycetemcomitans trxgene has 52 and 76% identities, respectively, to thetrxgenes ofEscherichia coliandHaemophilus influenzae. Enzymatic analysis revealed that the recombinant protein had the expected redox activity. When the recombinant thioredoxin was tested for its capacity to inhibit the production of cytokines by human peripheral blood mononuclear cells, it showed no significant inhibitory capacity. We therefore conclude that the thioredoxin ofA. actinomycetemcomitansdoes not act as an immunosuppressive factor, at least with human leukocytes in cultures, and that the identity of SF1 remains to be elucidated.
Enterotoxigenic
Escherichia coli
TibA Glycoprotein Adheres to Human Intestine Epithelial Cells
Authors: Lindenthal Christoph , Elsinghorst Eric A.
Published: July 2002

Enterotoxigenic
Escherichia coli
(ETEC) is capable of invading epithelial cell lines derived from the human ileum and colon. Two separate invasion loci (
tia
and
tib
) that direct noninvasive
E. coli
strains to adhere to and invade cultured human intestine epithelial cells have previously been isolated from the classical ETEC strain
H10407
. The
tib
locus directs the synthesis of TibA, a 104-kDa outer membrane glycoprotein. Synthesis of TibA is directly correlated with the adherence and invasion phenotypes of the
tib
locus, suggesting that this protein is an adhesin and invasin. Here we report the purification of TibA and characterization of its biological activity. TibA was purified by continuous-elution preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified TibA was biotin labeled and then shown to bind to HCT8 human ileocecal epithelial cells in a specific and saturable manner. Unlabeled TibA competed with biotin-labeled TibA, suggesting the presence of a specific TibA receptor in HCT8 cells. These results show that TibA acts as an adhesin. Polyclonal anti-TibA antiserum inhibited invasion of ETEC strain
H10407
and of recombinant
E. coli
bearing
tib
locus clones, suggesting that TibA also acts as an invasin. The ability of TibA to direct epithelial cell adhesion suggests a role for this protein in ETEC pathogenesis.